U.S. patent application number 13/394450 was filed with the patent office on 2013-01-10 for novel inhibitors of stearoyl-coa-desaturase-1 and their uses.
This patent application is currently assigned to CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE. Invention is credited to Zohra Benfodda, Lluis Fajas, Vanessa Fritz.
Application Number | 20130012709 13/394450 |
Document ID | / |
Family ID | 41568712 |
Filed Date | 2013-01-10 |
United States Patent
Application |
20130012709 |
Kind Code |
A1 |
Fajas; Lluis ; et
al. |
January 10, 2013 |
NOVEL INHIBITORS OF STEAROYL-CoA-DESATURASE-1 AND THEIR USES
Abstract
An inhibitor of the activity of stearoyl-CoA-desaturase-1
(SCD-1) enzyme for use in the treatment of prostate cancer as well
as novel inhibitors of formula (IIa).
Inventors: |
Fajas; Lluis; (Lausanne,
CH) ; Benfodda; Zohra; (Montpellier, FR) ;
Fritz; Vanessa; (Montpellier, FR) |
Assignee: |
CENTRE NATIONAL DE LA RECHERCHE
SCIENTIFIQUE
Paris
FR
|
Family ID: |
41568712 |
Appl. No.: |
13/394450 |
Filed: |
September 10, 2010 |
PCT Filed: |
September 10, 2010 |
PCT NO: |
PCT/IB10/54092 |
371 Date: |
September 19, 2012 |
Current U.S.
Class: |
544/365 ;
544/369; 544/370; 544/379; 544/383 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 277/56 20130101; C07D 333/38 20130101; C07D 213/82 20130101;
C07D 233/84 20130101; C07D 307/64 20130101; C07D 295/26 20130101;
C07D 213/81 20130101; C07D 333/34 20130101; A61K 31/495
20130101 |
Class at
Publication: |
544/365 ;
544/383; 544/379; 544/370; 544/369 |
International
Class: |
C07D 295/26 20060101
C07D295/26; C07D 403/12 20060101 C07D403/12; C07D 409/06 20060101
C07D409/06; C07D 401/06 20060101 C07D401/06; C07D 417/06 20060101
C07D417/06; C07D 409/12 20060101 C07D409/12; C07D 405/12 20060101
C07D405/12 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 2009 |
EP |
09290698.1 |
Claims
1. An inhibitor of an activity of stearoyl-CoA-desaturase-1 (SCD-1)
enzyme for treatment of prostate cancer, wherein said inhibitor of
activity of SCD-1 is a compound of formula (I): ##STR00220##
wherein: X represents --CO-- or --SO.sub.2--; R.sub.1 represents an
alkyl, a cycloalkyl, an aryl or a heteroaryl group in C.sub.5 to
C.sub.14, said aryl or heteroaryl being optionally substituted with
one or more groups R.sub.a; R.sub.a represents a halogen atom, a
hydroxyl group, --NO.sub.2, --CN, --NH.sub.2,
--N(C.sub.1-6alkyl).sub.2, a C.sub.1-6alkyl, a C.sub.1-6alkoxy, a
--C(O)--C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.3-6cycloalkyl,
aryl, C.sub.3-6 heterocyclyl or heteroaryl, said alkyl, alkoxy,
alkenyl, cycloalkyl, aryl, heterocyclyl or heteroaryl being
optionally substituted with one or more halogen atom, C.sub.1-6
alkyl, C.sub.1-6 alkoxy, --C(O)--C.sub.1-6 alkyl, --NO.sub.2,
--CF.sub.3, --OCF.sub.3, --CN, --NH.sub.2, and/or
--N(C.sub.1-6alkyl).sub.2; P is a heteroaromatic cycle in C.sub.5
to C.sub.14; x represents 0 or 1; Y represents --SO.sub.2-- or
*--CO--NH-- or *--CS--NH--, with * indicating a link to
--(P).sub.x--; n represents 0, 1, 2 or 3; R.sub.2 represents an
alkyl, a cycloalkyl, an aryl or a heteroaryl group in C.sub.5 to
C.sub.14, said aryl or heteroaryl being optionally substituted with
one or more groups R.sub.b; R.sub.b represents a halogen atom, a
hydroxyl group, NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, a
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, --C(O)--C.sub.1-6 alkyl,
--NH.sub.2, --N(C.sub.1-6alkyl).sub.2, C.sub.3-6 cycloalkyl,
C.sub.3-6 heterocyclyl, aryl, heteroaryl, said alkyl, alkoxy,
cycloalkyl, heterocyclyl, aryl, heteroaryl being optionally
substituted with one or more hydroxyl group, --CF.sub.3,
--OCF.sub.3, --NH.sub.2, --NO.sub.2, and/or --CN, or one of its
salts or enantiomer forms.
2. The inhibitor according to claim 1, wherein the prostate cancer
is a prostate cancer with a Gleason score equal or superior to
7.
3. The inhibitor according to claim 1, wherein X represents
--CO--.
4. The inhibitor according to claim 1, wherein R1 is an aryl or a
heteroaryl group in C.sub.5 to C.sub.14, said aryl or heteroaryl
being optionally substituted with one or more groups R.sub.a.
5. The inhibitor according to claim 1, wherein Y represents
--SO.sub.2-- or * --CO--NH--.
6. The inhibitor according to claim 1, wherein said compound is
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
] piperazine.
7. The inhibitor according to claim 1, wherein said inhibitor is of
formula (IIa): ##STR00221## wherein R.sub.1, R.sub.2 and n are as
defined in claim 1.
8. The inhibitor according to claim 1, wherein R.sub.1 represents a
phenyl group, optionally substituted with one or more R.sub.a, said
R.sub.a representing a halogen atom or a C.sub.1-6 alkoxy
group.
9. The inhibitor according to claim 1, wherein R.sub.1--(C.dbd.O)
represents a heteroarylcarbonyl group selected from the group
consisting of nicotinoyl, thiophene carbonyle, and
thiazolecarbonyle.
10. The inhibitor according to claim 1, wherein R.sub.2 is a
difluorophenyl group.
11. The inhibitor according to claim 1, wherein said inhibitor is
selected from the group consisting of:
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-methylphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-propylphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-isopropylphenyl)sulfonyl]piperazin-
e,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-nitrophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-chlorophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-bromophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4-[(4-fluorophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-methoxyphenyl]sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-acetylphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylmethane)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-biphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-fluorophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-t(3-fluorophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-trifluoromethylphenyl)sulfonyl]pip-
erazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-trifluoromethoxyphenyl)sul-
fonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-trifluoromethylphenyl)sulfonyl]pip-
erazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-trifluoromethylphenyl)sulf-
onyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2,6-difluorophenyl)sulfonyl]piper-
azine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-thiophene)sulfonyl]piperazin-
e,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-thiophene)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-furan)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-furan)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[1-methyl-1H-imidazole)sulfonyl]pipera-
zine,
1N-[(3-bromo-4-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]pi-
perazine,
1N-[(2-methoxy-5-bromobenzoyl)]-4N-[(2,4-difluorophenyl)sulfonyl-
]piperazine,
1N-[(2-methoxy-3-bromobenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperaz-
ine,
1N-[(4-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[(2-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[(3-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[nicotinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[isonicotinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[picolinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[3-thiophenecarbonyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[4-thiazolecarbonyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[(4-bromobenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
and their salts.
12. A compound of formula (IIa): ##STR00222## wherein: R.sub.1
represents an alkyl, an aryl or a heteroaryl group in C.sub.5 to
C.sub.14, said aryl or heteroaryl being optionally substituted with
one or more groups R.sub.a; R.sub.a represents a halogen atom, a
hydroxyl group, --NO.sub.2, --CN, --NH.sub.2,
--N(C.sub.1-6alkyl).sub.2, a C.sub.1-6alkyl, a C.sub.1-6alkoxy, a
--C(O)--C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.3-6cycloalkyl,
aryl, C.sub.3-6 heterocyclyl or heteroaryl, said alkyl, alkoxy,
alkenyl, cycloalkyl, aryl, heterocyclyl or heteroaryl being
optionally substituted with one or more halogen atom, C.sub.1-6
alkyl, C.sub.1-6 alkoxy, --C(O)--C.sub.1-6 alkyl, --NO.sub.2,
--CF.sub.3, --OCF.sub.3, --CN, --NH.sub.2, and/or
--N(C.sub.1-6alkyl).sub.2; R.sub.2 is a difluorophenyl group, and n
is 0, 1, 2, or 3.
13. A compound selected from the group consisting of: 1N
[(2-bromo-5-methoxybenzoyl)]-4N-[(phenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-methylphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-propylphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-isopropylphenyl)sulfonyl]piperazin-
e,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[4-nitrophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-chlorophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-bromophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4-[(4-fluorophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-methoxyphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-acetylphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylmethane)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-biphenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-fluorophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-fluorophenyl)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-trifluoromethylphenyl)sulfonyl]pip-
erazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-trifluoromethoxyphenyl)sul-
fonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-trifluoromethylphenyl)sulfonyl]pip-
erazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-trifluoromethylphenyl)sulf-
onyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2,6-difluorophenyl)sulfonyl]piper-
azine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-thiophene)sulfonyl]piperazin-
e,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-thiophene)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-furan)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-furan)sulfonyl]piperazine,
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(1-methyl-1H-imidazole)sulfonyl]piper-
azine,
1N-[(3-bromo-4-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]p-
iperazine, 1N-[(2-methoxy-5-bromobenzoyl)]-4N-[(2,4-di
fluorophenyl) sulfonyl] piperazine,
1N-[(2-methoxy-3-bromobenzoyl)]-4-N-[(2,4-di
fluorophenyl)sulfonyl]piperazine,
1N-[(4-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[(2-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[(3-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[nicotinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[isonicotinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[picolinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[3-thiophenecarbonyl]-4-N-[(2,4-difluorophenyl)
sulfonyl]piperazine,
1N-[4-thiazolecarbonyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
1N-[(4-bromobenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
and their salts.
14. A pharmaceutical composition containing as an active agent at
least one compound according to claim 12.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the therapy of cancers and
more specifically of prostate cancer (PC). The present invention
more precisely deals with the use of some piperazine derivatives
for their activity against prostate cancer cells.
BACKGROUND OF THE INVENTION
[0002] Prostate cancer (PC) is the most commonly diagnosed cancer
and the second leading cause of cancer death in western men after
middle-age, and remained a major research and public health
priority.
[0003] With about 40,000 new cases and 10,000 deaths per year in
France, PC has remained a major research and public health
priority. Consistent with the trend towards an aging global
population, the incidence of the disease is increasing worldwide to
an average rate of 3% a year, and affects 10% of subjects beyond 70
years of age.
[0004] Bone is the main site of metastasis in PC, and up to 90% of
affected patients in later stage develop skeletal metastases that
are associated with significant morbidity and mortality. With
almost two millions men battling prostate cancer today and the
three million more predicted in the next decade, interventions to
prevent, treat and avoid prostate cancer and its complications are
of great economic and social impact.
[0005] Among men with early, organ-confined, PC, primary treatment
with radical prostatectomy or radiotherapy in patients demonstrates
overall 10-year survival rates of over 75%. However, it is
estimated that approximately 40% of patients will relapse after
definitive local therapy. In these patients, the use of additional
therapies is required.
[0006] Because the growth of PC is initially androgen-dependent,
numbers of pharmaceutical groups have been positioned in androgen
deprivation therapeutic approach. Thus, over 90% of patients who
failed radiotherapy undergo androgen deprivation therapy (ADT).
Thus, a number of pharmaceutical groups are positioned in
therapeutic approaches attempt to interrupt the production and/or
the action of testosterone, with the development of Luteinizing
hormone-releasing hormone (LHRH) agonists, such as Leuprolide
(Bayer AG, Sanofi-Aventis, TAP Pharmaceuticals), Goserelin
(AstraZeneca), Triptorelin (Ipsen, Ferring Pharmaceuticals,
Pfizer), or to block the action of testosterone by using
anti-androgens, such as Bicalutamide (AstraZeneca) and Flutamide
(Schering-Plough).
[0007] However, despite an initial response to ADT, ultimately all
patients with advanced PC experience disease progression and
invariably become refractory to hormonal manipulation within 18-24
months. Historically, once men reach this stage, therapeutic
options are limited and prognosis is poor, with a median survival
time of 12-16 months. Moreover, up to 90% of patients with advanced
PC are affected by skeletal metastasis, an incurable progression of
the disease that accounts for the vast majority of disease-related
mortality and its associated morbidity. Although systemic therapy
has proven effective for many cancers, currently used
chemotherapeutic agents are not curative in the treatment of
androgen-independent prostate cancer (AIPC). Indeed, no single
chemotherapeutic agent such as Mitoxantrone (Merk), Docetaxel
(Sanofi-Aventis), Ixabepilone (Bristol-Myers Squibb), or
combination of agents with corticosteroids, has been shown to
prolong life.
[0008] Nevertheless, despite an initial response to treatment, most
patients with advanced disease eventually develop resistance and
progress to hormone refractory PC for which there is currently no
curative therapy. Although many efforts have been directed towards
more efficient ways to ablate the androgen action, little attention
has been paid to the metabolism of prostate cancer cells.
[0009] In view of this, there is currently no curative treatment of
prostate cancer and new therapeutic options are expected.
SUMMARY OF THE INVENTION
[0010] The present invention raises from the unexpected observation
of the inventors that inhibitors of SCD-1 activity are potent new
therapeutic agent to inhibit PC disease.
[0011] High ratio of monounsaturated to saturated fatty acids
affect phospholipids composition and has been correlated with
neoplastic transformation, as demonstrated by FA analysis of lipids
extracts from transformed cells, cancerous cells and tumor tissue
(Apostolov et al., Blut, 1985, 50:349; Wood et al., Eur. J. Surg.
Oncol., 1985, 11:347). Moreover, studies have evidenced that
stearic acid treatment leads to inhibition of tumor cell growth
both in vitro and in vivo (Habib et al., Br. J. Cancer, 1987,
56:455).
[0012] Unexpectedly, the inventors have observed that inhibition of
SCD-1 activity altered lipid synthesis and proliferation in
prostate cancer cell lines.
[0013] In addition, as shown in the following examples, by using
siRNA, they observe that specific silencing hSCD-1 gene expression
significantly reduces proliferation in both androgen-sensitive
LNCaP and androgen-insensitive C4-2 prostate cancer cell lines.
[0014] Consequently, in one aspect, the present invention relates
to inhibitors of the expression and/or activity of human
stearyl-CoA-desaturase (hSCD) enzymes, especially of
stearyl-CoA-desaturase-1 (SCD-1) for use in the treatment of
prostate cancer.
[0015] Accordingly to one embodiment, the prostate cancer is a
prostate cancer with a Gleason score equal or superior to 7.
[0016] According to one embodiment, the instant invention relates
to the use of inhibitors of the expression of SCD-1.
[0017] According to another embodiment, the instant invention
relates to the use of inhibitors of the expression of SCD-1
selected from the group consisting of antisense RNA or DNA
molecules, small interfering RNAs (siRNAs), short hairpin RNAs
(shRNAs) and ribozymes.
[0018] According to another embodiment, the instant invention
relates to the use of inhibitors of the activity of SCD1.
[0019] According to another embodiment, the instant invention
relates to the use of inhibitors of the activity of SCD1 of formula
(I):
##STR00001##
[0020] wherein X, R.sub.1, P, x, Y, n and R.sub.2 are as defined
hereafter.
[0021] According to another aspect, the instant invention relates
to inhibitors of the expression and/or activity of human
stearyl-CoA-desaturase (hSCD) enzymes, especially
stearyl-CoA-desaturase-1 (SCD-1), and in particular of compounds of
the formula (I), for use as selective agent for blocking prostate
cancer (PC) cells proliferation
[0022] The present invention also relates to a use of an inhibitor
of the expression and/or activity of human stearyl-CoA-desaturase
(hSCD) enzymes, especially stearyl-CoA-desaturase-1 (SCD-1), and in
particular of compounds of the formula (I), as selective agent for
blocking prostate cancer (PC) cells proliferation for the
preparation of a pharmaceutical composition for treating prostate
cancer.
[0023] In another aspect, the invention relates to a method for
treating a prostate cancer, comprising the administration to a
patient in need thereof of an effective amount of at least one
inhibitor of the expression and/or activity of human SCD enzymes,
especially SCD-1, and in particular of compounds of the formula
(I).
[0024] According to another aspect, the invention relates to
compounds of formula (II) as defined hereafter.
[0025] In a another aspect, the invention relates to a
pharmaceutical composition comprising an effective amount of at
least a compound of formula (II) as defined hereafter, optionally
in combination with at least one cancer agent different from said
compound of formula (II), and in particular a secondary
chemotherapeutic agent.
[0026] According to another aspect, the present invention relates
to a pharmaceutical composition containing as active agent at least
one compound according to the invention, and in particular of
formula (IIa) as defined hereafter.
[0027] A pharmaceutical composition of the invention comprises an
active compound in a therapeutically effective amount.
[0028] Such compound of formula (II) and/or pharmaceutical
composition containing at least one of them are particularly useful
for preventing and/or treating SCD-mediated disease. These diseases
are detailed hereafter.
[0029] Due to its role in lipid metabolism, SCD-1 is a therapeutic
target for the treatment of diseases related to metabolic syndrome,
including but not limited to cancer, acnea, obesity and
obesity-related diseases, that is hypertension, insulin resistance,
diabetes, atherosclerosis and heart failure.
[0030] An SCD-mediated disease or condition includes metabolic
syndrome (including but not limited to dyslipidemia, obesity and
insulin resistance, hypertension, microalbuminemia, hyperuricaemia,
and hypercoagulability), Syndrome X, diabetes, insulin resistance,
decreased glucose tolerance, non-insulin-dependent diabetes
mellitus, Type II diabetes, Type I diabetes, diabetic
complications, body weight disorders, weight loss, body mass index
and leptin related diseases.
[0031] An SCD-mediated disease or condition also includes fatty
liver, hepatic steatosis, hepatitis, non-alcoholic hepatitis,
non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute
fatty liver, fatty liver of pregnancy, drug-induced hepatitis,
erythrohepatic protoporphyria, iron overload disorders, hereditary
hemochromatosis, hepatic fibrosis, hepatic cirrhosis, hepatoma and
conditions related thereto.
[0032] An SCD-mediated disease or condition also includes but is
not limited to a disease or condition which is, or is related to
primary hypertriglyceridemia, or hypertriglyceridemia secondary to
another disorder or disease, such as hyperlipoproteinemias,
familial histiocytic reticulosis, lipoprotein lipase deficiency,
apolipoprotein deficiency (such as ApoC II deficiency or ApoE
deficiency), and the like, or hypertriglyceridemia of unknown or
unspecified etiology.
[0033] An SCD-mediated disease or condition also includes a
disorder of polyunsaturated fatty acid (PUFA) disorder, or a skin
disorder, including but not limited to eczema, acne, psoriasis,
keloid scar formation or prevention, diseases related to production
or secretions from mucous membranes, such as monounsaturated fatty
acids, wax esters, and the like. An SCD-mediated disease or
condition also includes inflammation, sinusitis, asthma,
pancreatitis, osteoarthritis, rheumatoid arthritis, cystic
fibrosis, and pre-menstrual syndrome.
[0034] An SCD-mediated disease or condition also includes but is
not limited to a disease or condition which is, or is related to
cancer, more particularly lung cancer and prostate cancer, breast
cancer, hepatomas and the like, neoplasia, malignancy, metastases,
tumours (benign or malignant), carcinogenesis.
[0035] An SCD-mediated disease or condition also includes a
condition where increasing lean body mass or lean muscle mass is
desired, such as is desirable in enhancing performance through
muscle building. Myopathies and lipid myopathies such as carnitine
palmitoyltransferase deficiency (CPT I or CPT II) are also included
herein. Such treatments are useful in humans and in animal
husbandry, including for administration to bovine, porcine or avian
domestic animals or any other animal to reduce triglyceride
production and/or provide leaner meat products and/or healthier
animals.
[0036] An SCD-mediated disease or condition also includes a disease
or condition which is, or is related to, neurological diseases,
psychiatric disorders, multiple sclerosis, eye diseases, and immune
disorders.
DEFINITIONS
[0037] "Therapeutically effective amount" refers to an amount of a
compound of the invention which, when administered to a mammal,
preferably a human, is sufficient and necessary to effect a
treatment, as defined below, in the mammal, preferably a human. The
amount of a compound of the invention which constitutes a
"therapeutically effective amount" will vary depending on the
compound, the condition and its severity, and the age of the mammal
to be treated, but can be determined routinely by one of ordinary
skill in the art having regard to his own knowledge and to this
disclosure.
[0038] "Treating" or "treatment" as used herein covers the
treatment of the disease or condition of interest in a mammal,
preferably a human, having the disease or disorder of interest, and
includes: [0039] (i) preventing the disease or condition from
occurring in a mammal, in particular, when such mammal is
predisposed to the condition but has not yet been diagnosed as
having it; [0040] (ii) inhibiting the disease or condition, i.e.,
arresting its development; or [0041] (iii) relieving the disease or
condition, i.e., causing regression of the disease or
condition.
[0042] The "prophylactic and/or therapeutic agent" as hereunder
mentioned may be a compound according to the invention itself
having a prophylactic and/or therapeutic action on indicated
diseases or a pharmaceutical agent containing such a substance.
[0043] As used herein, the terms "disease" and "condition" may be
used interchangeably or may be different in that the particular
malady or condition may not have a known causative agent (so that
etiology has not yet been worked out) and it is therefore not yet
recognized as a disease but only as an undesirable condition or
syndrome, wherein a more or less specific set of symptoms have been
identified by clinicians.
[0044] According to the present invention, the terms below have the
following meanings
[0045] The term "halogen atom" corresponds to a fluorine, chlorine,
bromine or iodine atom.
[0046] The term "alkyl" as used herein refers to a saturated,
linear or branched aliphatic group. The following examples may be
cited: methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,
2-methyl-1-propyl (also named i-Bu), 2-butyl (also named s-Bu),
2-methyl-2-propyl (also named t-Bu), 1-pentyl (also named
n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,
3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl,
2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,
3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,
3,3-dimethyl-2-butyl, n-pentyl, n-hexyl. Preferred alkyl according
to the invention are methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,
2-methyl-1-propyl (also named i-Bu), 2-butyl (also named s-Bu),
2-methyl-2-propyl (also named t-Bu), 1-pentyl (also named
n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,
3-methyl-1-butyl, 2-methyl-1-butyl.
[0047] As used herein and unless otherwise stated, the term
"cycloalkyl" means a saturated cyclic alkyl group as defined above.
The following examples may be cited: cyclopropyl,
methylcyclopropyl, cyclobutyl, methylcyclobutyl, methylcyclopentyl,
cyclopentyl, cyclohexyl. Preferred cycloalkyl according to the
invention are cyclopentyl or cyclohexyl.
[0048] The term "alkenyl" corresponds to a linear or branched,
unsaturated aliphatic group, comprising at least one unsaturation
site (usually 1 to 3 and preferably 1), i.e. a carbon-carbon sp2
double bound. The following examples may be cited: ethylene, allyl.
The double bond may be in the cis or trans configuration.
[0049] The term "alkoxy" corresponds to a --O-alkyl group, wherein
the alkyl group is as defined above. The following examples may be
cited: methoxy, ethoxy, propoxy.
[0050] The term "aryl" as used herein means an aromatic mono- or
poly-cyclic group in C.sub.5 to C.sub.14. An example of monocyclic
group may be phenyl. Examples of polycyclic rings may be
naphthalene, anthracene, and biphenyl.
[0051] The term "heterocyclyl" or "heterocycloalkyl" as used herein
refers to a cycloalkyl in C.sub.5 to C.sub.14 as described above
further comprising at least one heteroatom chosen from nitrogen
(N), oxygen (O), or sulphur (S) atom. The following examples may be
cited: piperidinyl, piperazinyl, morpholinyl, 1,4-dioxanyl,
1,4-dithianyl, homomorpholinyl, 1,3,5-trithianyl, pyrrolidinyl,
2-pyrrolidinyl, tetrahydro furanyl, tetrahydropyranyl.
[0052] The term "heteroaryl" as used herein corresponds to an
aromatic, mono- or poly-cyclic group comprising between 5 and 14
carbon atoms and comprising at least one heteroatom such as
nitrogen, oxygen or sulphur atom. Examples of such mono- and
poly-cyclic heteroaryl group may be: pyridyl, thiazolyl,
thiophenyl, furanyl, pyranyl, pyrrolyl, pyrazolyl, imidazolyl,
tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, quinolinyl,
isoquinolinyl, benzimidazolyl, pyrrolinyl, isothiazolyl,
isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, imidazo
linyl, pyrazo lidinyl, pyrazo linyl, indolinyl, iso indo linyl,
oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl,
benzoxazolinyl, benzothienyl, benzothiazolyl, pyridinyl,
dihydropyridyl, pyrimidinyl, pyrazinyl, oxazolyl, thiofuranyl
(thiophenyl or thienyl).
[0053] Rings as defined above, comprising at least one heteroatom,
may be bound through a carbon atom or a heteroatom.
[0054] Regardless of bond indications, if a substituent is
polyvalent (based on its position in the structure referred to,
then any and all possible orientations of the substituent are
intended.
DETAILED DESCRIPTION OF THE INVENTION
[0055] The compounds considered according to the invention as
active against the PC are capable of inhibiting the expression
and/or activity of the human SCD-1 enzyme.
[0056] According to one embodiment, an inhibitor preferably
considered in the invention may be an inhibitor of the expression
of SCD-1.
[0057] In another preferred embodiment, an inhibitor of the
expression of the SCD-1 more particularly considered in the
invention may be selected from the group consisting of antisense
RNA or DNA molecules, small interfering RNAs (siRNAs), short
hairpin RNAs (shRNAs) and ribozymes.
[0058] RNA interference is a post-transcriptional process observed
in various organisms whereby double-stranded RNA molecules mediate
gene silencing in a sequence-specific manner. RNA interference may
be carried out using short interfering RNAs (siRNAs), which are
generally about 19-22 nucleotides long.
[0059] As example of siRNA that may be used in the instant
invention one may mention SEQ ID NO: 1 GCACAUCAACUUCACCACA.
[0060] A small hairpin RNA or short hairpin RNA (shRNA) is a
sequence of RNA that makes a tight hairpin turn that can be used to
silence gene expression via RNA interference.
[0061] Antisense polynucleotides (such as antisense RNAs or DNAs)
may be used to suppress expression of a gene, either before or
after transcription. Antisense polynucleotide is a polynucleotide
which contains a stretch of nucleotides complementary to another
polynucleotide (RNA or DNA) that has some cellular function. The
length of the complementary stretch is usually a few hundred
nucleotides, but shorter stretches can also be used.
[0062] As examples of method useful in the invention for obtaining
antisense RNAs or antisense DNAs, siRNA or shRNAs, one may mention
the methods described in WO 2006/060454, WO 2001/025488, WO
2004/108897, or US 2006/223777.
[0063] Ribozymes are synthetic RNA molecules having highly specific
endoribonuclease activity. A ribozyme comprises a hybridizing
region which is complementary in nucleotide sequence to at least
part of a target RNA, and a catalytic region which is adapted to
cleave the target RNA. Methods that may be used for obtaining
ribozymes in accordance with the invention are, for example,
described in U.S. Pat. No. 5,494,814 or EP 0 321 201.
[0064] On the basis of the nucleic acid sequence encoding for the
human SCD-1 (NCBI Reference Sequence NM.sub.--005063.4) and on its
knowledge in the field, a man skilled in the art may readily obtain
antisense RNA or DNA molecules, small interfering RNAs (siRNAs),
short hairpin RNAs (shRNAs) and ribozymes suitable for the
invention.
[0065] According to another embodiment, an inhibitor preferably
considered in the invention may be an inhibitor of the activity of
SCD-1.
[0066] The determination of inhibiting the activity of human SCD1
enzyme may be readily accomplished using the SCD-1 enzyme and
microsomal assay procedure described in Brownlie et al., (WO
01/62954).
[0067] More particularly these SCD-1 inhibitors may be selected
from the group consisting of: [0068] pyridine derivatives as
disclosed in WO 2005/011654 and WO 2005/011656 and in particular
the following compounds,
[0068] ##STR00002## [0069] pyridazines derivatives as disclosed in
WO 2005/011655 and WO 2006/086447 and in particular the following
compound,
[0069] ##STR00003## [0070] piperidine derivatives as the following
compound,
[0070] ##STR00004## [0071] nicotinamides derivatives as disclosed
in WO 2006/014168, [0072] heterocyclic bicyclic derivatives as
disclosed in WO 2006/034312 and in particular the following
compound,
[0072] ##STR00005## [0073] heterocyclic derivatives as disclosed in
WO 2006/034315, WO 2006/034338, WO 2006/034446, WO 2006/101521 and
WO 2006/034441, and in particular the following compounds,
[0073] ##STR00006## [0074] piperazines derivatives as disclosed in
WO 2006/125180, and in particular the following compounds,
[0074] ##STR00007## [0075] heteroaryl derivatives as disclosed in
WO 2007/044085, and in particular the following compound,
[0075] ##STR00008## [0076] thiazolidines derivatives as disclosed
in WO 2007/046868, and in particular the following compound,
[0076] ##STR00009## [0077] piperidines derivatives as disclosed in
WO 2007/050124, and in particular the following compound,
[0077] ##STR00010## [0078] aza cyclohexane derivatives as disclosed
in WO 2007/056846, and in particular the following compound,
[0078] ##STR00011## [0079] heteroaromatic derivatives as disclosed
in WO 2007/071023 and in the publication from J. Med. Chem., 2007,
50, 13, 3086-3100, and in particular the following compounds,
[0079] ##STR00012## [0080] bicyclic heteroaromatic derivatives as
disclosed in WO 2009/012573, and in particular the following
compounds,
##STR00013##
[0081] According to another preferred embodiment, an inhibitor of
activity of the SCD-1 may be a compound represented by the
following formula (I):
##STR00014##
wherein [0082] R.sub.1 represents an alkyl, a cycloalkyl, an aryl
or a heteroaryl group in C.sub.5 to C.sub.14, in particular in
C.sub.6, said aryl or heteroaryl being optionally substituted with
one or more groups R.sub.a; [0083] R.sub.a represents an halogen
atom, an hydroxyl group, --NO.sub.2, --CN, --NH.sub.2,
--N(C.sub.1-6alkyl).sub.2, a C.sub.1-6alkyl, a C.sub.1-6alkoxy, a
--C(O)--C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.3-6cycloalkyl,
aryl, C.sub.3-6 heterocyclyl or heteroaryl, said alkyl, alkoxy,
alkenyl, cycloalkyl, aryl, heterocyclyl or heteroaryl being
optionally substituted with one or more halogen atom, C.sub.1-6
alkyl, C.sub.1-6 alkoxy, --C(O)--C.sub.1-6 alkyl, --NO.sub.2,
--CF.sub.3, --OCF.sub.3, --CN, --NH.sub.2, and/or
--N(C.sub.1-6alkyl).sub.2; [0084] P is a heteroaromatic cycle in
C.sub.5 to C.sub.14, in particular in C.sub.6; [0085] x represents
0 or 1; [0086] Y represents --SO.sub.2-- or *--CO--NH--,
*--CS--NH--, with * figuring the link to --(P).sub.x--; [0087] n
represents 0, 1, 2 or 3; [0088] R.sub.2 represents an alkyl, a
cycloalkyl, an aryl or a heteroaryl group in C.sub.5 to C.sub.14,
in particular in C.sub.6, said aryl or heteroaryl being optionally
substituted with one or more groups R.sub.b; [0089] R.sub.b
represents an halogen atom, an hydroxyl group, NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, a C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
--C(O)--C.sub.1-6 alkyl, --NH.sub.2, --N(C.sub.1-6alkyl).sub.2,
C.sub.3-6 cycloalkyl, C.sub.3-6 heterocyclyl, aryl, heteroaryl,
said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl
being optionally substituted with one or more hydroxyl group,
--CF.sub.3, --OCF.sub.3, --NH.sub.2, --NO.sub.2, and/or --CN, or
one of its salts or enantiomer forms.
[0090] The compounds of formula (I) can comprise one or more
asymmetrical carbon atoms. They can thus exist in the form of
enantiomers or of diastereoisomers. These enantiomers,
diastereoisomers, or their mixtures, including the racemic mixtures
form part of the invention.
[0091] The compounds of the invention may exist in the form of free
bases or of addition salts with pharmaceutically acceptable
acids.
[0092] Suitable pharmaceutically acceptable salts of compounds of
formula (I) include base addition salts and where appropriate acid
addition salts. Suitable physiologically acceptable addition salts
of compounds (I) include alkali metal or alkaline metal salts such
as sodium, potassium, calcium, and magnesium salts, and ammonium
salts, formed with amino acids (e.g. lysine and arginie) and
organic bases (e.g. procaine, phenylbenzylamine, ethanolamine
diethanolamine and N-methyl glucosamine).
[0093] Suitable acid addition salts may be formed with organic acid
and inorganic acids, e.g. hydrochloric acid.
[0094] The compounds of formula (I) can also exist in the form of a
hydrate or of a solvate, i.e. in the form of associations or
combinations with one or more water or solvent molecules. Such
hydrates and solvates also form part of the invention.
[0095] According to a specific embodiment in the above-defined
compounds of formula (I) of the present invention, X represents
--CO--.
[0096] According to another specific embodiment in the
above-defined compounds of formula (I) of the present invention, R1
is an aryl or a heteroaryl group in C.sub.5 to C.sub.14, in
particular in C.sub.6, said aryl or heteroaryl being optionally
substituted with one or more groups R.sub.a.
[0097] According to another specific embodiment in the
above-defined compounds of formula (I) of the present invention, Y
represents --SO.sub.2-- or *--CO--NH--, with * figuring the link to
--(P).sub.x--.
[0098] According to a preferred variant, an SCD inhibitor may be
the
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[phenylpropan)pyridine-3-carboxamide]p-
iperazine (BZ36).
##STR00015##
or one of its salts or enantiomer forms.
[0099] As shown hereafter, treatment with the
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine significantly inhibits the growth of PC cell lines both
in vitro and in vivo in subcutaneous xenografts mouse models of
PC.
[0100] According to another preferred variant, a compound
considered according to the invention may be of general formula
(II):
##STR00016##
[0101] wherein X, Y, R.sub.1, R.sub.a, R.sub.2, R.sub.b and n are
as defined previously.
[0102] The compounds of formula (II) form also part of the present
invention as new compounds.
[0103] More particularly, the compound of formula (II) may be
represented by one of the following formula:
##STR00017##
[0104] with R.sub.1, n and R.sub.2 being as defined previously.
[0105] Among the compound of formula (IIa) to (IIi) here-above
defined, mention may be more particularly made to the compounds of
formula (IIa).
[0106] A first class of compounds of formula (IIa) is defined with
R.sub.1 representing a phenyl group, optionally substituted with
one or more R.sub.a, said R.sub.a representing an halogen atom or a
C.sub.1-6alkoxy group, and more particularly a bromo, methoxy a
benzoyl group; and n represents 0 or 1.
[0107] According to a specific embodiment, R.sub.1 is a
2-bromo-5-methoxybenzoyl group.
[0108] A second class of compounds of formula (IIa) is defined with
R.sub.1--(C.dbd.O) representing a heteroarylcarbonyl group selected
form the group consisting of nicotinoyl, thiophene carbonyle, and
thiazolecarbonyle.
[0109] According to a specific embodiment in the above-defined
compounds of formula (II) and (IIa), R.sub.2 may preferably be a
difluorophenyl group.
[0110] According to another specific embodiment in the
above-defined compounds of formula (IIa):
[0111] R.sub.1 represents an alkyl, an aryl or a heteroaryl group
in C.sub.5 to C.sub.14, in particular in C.sub.6, said aryl or
heteroaryl being optionally substituted with one or more groups
R.sub.a;
[0112] R.sub.a represents an halogen atom, an hydroxyl group,
--NO.sub.2, --CN, --NH.sub.2, --N(C.sub.1-6alkyl).sub.2, a
C.sub.1-6alkyl, a C.sub.1-6alkoxy, a --C(O)--C.sub.1-6alkyl,
C.sub.2-6alkenyl, C.sub.3-6cycloalkyl, aryl, C.sub.3-6 heterocyclyl
or heteroaryl, said alkyl, alkoxy, alkenyl, cycloalkyl, aryl,
heterocyclyl or heteroaryl being optionally substituted with one or
more halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
--C(O)--C.sub.1-6 alkyl, --NO.sub.2, --CF.sub.3, --OCF.sub.3, --CN,
--NH.sub.2, and/or --N(C.sub.1-6alkyl).sub.2;
[0113] R.sub.2 is a difluorophenyl group, and
[0114] n is as defined above in formula (I).
[0115] More particularity, the compounds of formula (II) considered
according to the invention may be group consisting of: [0116]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenyl)sulfonyl]piperazine,
[0117]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-methylphenyl)sulfonyl]piperazine,
[0118]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-propylphenyl)sulfonyl]piper-
azine, [0119]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-isopropylphenyl)sulfonyl]piperazin-
e, [0120]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-nitrophenyl)sulfonyl]pipe-
razine, [0121]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-chlorophenyl)sulfonyl]piperazine,
[0122]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-bromophenyl)sulfonyl]pipera-
zine, [0123]
1N-[(2-bromo-5-methoxybenzoyl)]-4-[(4-fluorophenyl)sulfonyl]piperazine,
[0124]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-methoxyphenyl)sulfonyl]pipe-
razine, [0125]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-acetylphenyl)sulfonyl]piperazine,
[0126]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylmethane)sulfonyl]pipera-
zine, [0127]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-biphenyl)sulfonyl]piperazine,
[0128]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-fluorophenyl)sulfonyl]piper-
azine, [0129]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-fluorophenyl)sulfonyl]piperazine,
[0130]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-trifluoromethylphenyl)sulfo-
nyl]piperazine, [0131]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-trifluoromethoxyphenyl)sulfonyl]pi-
perazine, [0132]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-trifluoromethylphenyl)sulfonyl]pip-
erazine, [0133]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-trifluoromethylphenyl)sulfonyl]pip-
erazine, [0134]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne, [0135]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2,6-difluorophenyl)sulfony-
l]piperazine, [0136]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-thiophene)sulfonyl]piperazine,
[0137]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-thiophene)sulfonyl]piperazi-
ne, [0138]
1N-[1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-furan)sulfonyl]piper-
azine, [0139]
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-furan)sulfonyl]piperazine,
[0140] 1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(1-methyl-1H-imidazo
le)sulfonyl]piperazine, [0141]
1N-[(3-bromo-4-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperaz-
ine, [0142]
1N-[(2-methoxy-5-bromobenzoyl)]-4N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne, [0143]
1N-[(2-methoxy-3-bromobenzoyl)]-4-N-[(2,4-difluorophenyl)sulfon-
yl]piperazine, [0144]
1N-[(4-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
[0145]
1N-[(2-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne, [0146]
1N-[(3-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piper-
azine, [0147]
1N-[nicotinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
[0148]
1N-[isonicotinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
[0149]
1N-[picolinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
[0150]
1N-[3-thiophenecarbonyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine,
[0151]
1N-[4-thiazolecarbonyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne, [0152]
1N-[(4-bromobenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperaz-
ine, and their salts.
[0153] Compounds pertaining to the hereabove defined formula (IIa)
form part of the invention as the pharmaceutical composition
including them.
[0154] More specifically, the instant invention is also more
particularly directed to the compound examples described in the
following examples 3, 8, 9, 11, 12, 13 16, 17, 21, 23, 26, 28.
[0155] The compounds considered according to the invention may be
easily obtained according to the following scheme for
synthesis:
##STR00018##
with R.sub.1 and R.sub.2 being as defined previously.
[0156] To a solution of (A) (1 equiv) in anhydrous solvent like for
example CH.sub.2Cl.sub.2, substituted phenyl sulfonyl chloride (1
equiv) is added in presence of a base like, for example, N,N
diisopropyl ethyl amine. The mixture was maintained under stirring
until the expected reaction be completed. The compound (B) is
isolated from the rectional mixture according to a conventional
method.
[0157] The starting compounds are generally commercially available
or can be prepared according to methods known to the person skilled
in the art in particular as disclosed in the following example.
[0158] The following examples provide with details of preparation
of compounds according to the invention.
EXPERIMENTAL SECTION
[0159] Reagents were obtained from Sigma-Aldrich or Acros.
Characterization of all compounds was done with .sup.1H, .sup.19F,
.sup.13C NMR, and HRMS. .sup.1H, .sup.19F, and .sup.13C NMR spectra
were recorded at 300.13 MHz, 282.37 MHz and 75.46 MHz respectively
with a Brucker Avance 300 spectrometer, therefore chemical shifts
were given in ppm relative to Me.sub.4Si, CCl.sub.3F respectively,
as internal standards. Coupling constants were given in Hz. High
Resolution Mass Spectrometry (HRMS) were recorded on a Jeol SX 102
spectrometer. High Pressure Liquid Chromatographie (HPLC) analyses
were obtained on the Waters Alliance 2795 using the following
conditions: thermo Hypersil C18 column (3 .mu.m, 50 mm L.times.2.1
mm ID), 20.degree. C. column temperature, 0.2 mL/min flow rate,
photodiodearray detection (210-400 nm).
[0160] Melting points were recorded at atmospheric pressure unless
otherwise stated on a Stuart scientific SMP3 apparatus and were
remained without any correction. The products were purified by
column chromatography.
[0161] Thin layer chromatography was performed with Merck Silica
gel aluminium-backed plate with UV visualization. The following
synthetic conditions have not been optimized.
Preparation of 1N-(2-bromo-5-methoxybenzoyl)piperazine
[0162] In a 1000 mL flask were added 370 mL of CH.sub.2Cl.sub.2 and
12.63 g (147 mmol) of piperazine. The resulting solution,
maintained at 0.degree. C. using an ice bath, was added dropwise a
ditert butyldicarbonate solution (16 g, 73.5 mmol in 150 mL of
CH.sub.2Cl.sub.2). The mixture was stirred for additionnal 1 h,
filtered and the filtrate concentrated to dryness. Water (220 mL),
was added to the resulting oil and the mixture filtered. The
filtrate was saturated with potassium carbonate and extracted with
diethyl ether (3.times.100 mL). The solvent was dried over
Na.sub.2SO.sub.4 and concentrated to dryness yielding to 9 g
tert-butyl piperazine-1-carboxylate (66%).
[0163] A solution of so-obtained piperazine-1-carboxylic acid
tert-butyl ester (8.7 g, 47 mmol) and N,N-diisopropyl ethyl amine
(13.6 mL, 78 mmol) in CH.sub.2Cl.sub.2 at 0.degree. C. was added
2-bromo-5-methoxybenzoyl chloride (12.96 g, 52 mmol). The mixture
was allowed to warm to room temperature and stirred for 2 h then
poured into water and extracted with CH.sub.2Cl.sub.2. The organic
layer was dried over anhydrous Na.sub.2SO.sub.4 and concentrated
under reduced pressure. The crude material was purified by column
chromatography (Petroleum ether/Ethyl acetate 70:30) to give
compound (16 g, 85%). R.sub.f. 0.25 (Petroleum ether/Ethyl acetate
70:30).
[0164] Further, trifluoroacetic acid (15 mL) was added slowly at
0-5.degree. C. under nitrogen to a solution of
tert-butyl-4-(2-bromo-5-methoxybenzoyl)piperazine-1-carboxylate (16
g, 40 mmol) in CH.sub.2Cl.sub.2 (15 mL). The mixture was stirred at
room temperature for 2 h then poured on to cold water (50 mL). The
aqueous solution was made basic by the addition of NaOH (1N), then
the product was extracted into CH.sub.2Cl.sub.2 (2.times.100 mL).
The combined extracts were washed with brine (200 mL), dried over
anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure
yielding to 10.2 g (85%) of the title compound.
Preparation of 4N-(2,4-difluorophenylsulfonyl)piperazine
[0165] To a solution of piperazine-1-carboxylic acid tert butyl
ester (4.37 g, 23.5 mmol) and N,N-diisopropyl ethyl amine (6.14 mL,
35.3 mmol) in CH.sub.2Cl.sub.2 at 0.degree. C. was added
2,4-difluorophenylsulfonyl chloride (5 g, 23.5 mmol). The mixture
was allowed to warm to room temperature and stirred for 2 h then
poured into water and extracted with CH.sub.2Cl.sub.2. The crude
material was used in the next step without any purification. The
solvent was dried over Na.sub.2SO.sub.4 and concentrated to dryness
yielding to 8 g of the title compound (94%). Then the product,
sudden a remplacement of the boc grouping according to higher
described protocol, we so obtain a new reagent of the general
synthesis.
Example 1
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenyl)sulfonyl]piperazine
[0166] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and phenylsulfonyl chloride (0.21 mL, 1.67 mmol) and
N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.47 g, 64%).
[0167] mp: 180.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.75 (m, 1H), 2.9 (m, 3H), 3.05 (m, 1H), 3.15 (m, 1H), 3.55 (s,
OCH.sub.3), 3.6 (m, 1H), 3.75 (m, 1H), 6.4 (d, J=3 Hz, 1H), 6.55
(dd, J=8.8 Hz, 3 Hz, 1H), 7.15 (dd, J=8.8 Hz, 1H), 7.3-7.5 (m, 3H),
7.65 (d, J=4.96 Hz, 2H); .sup.13C NMR (75.46 MHz, CDCl3): .delta.
38.8, 43.5, 43.9, 43.9, 53.5, 106.9, 110.9, 114.5, 125.5 (2C),
127.1 (2C), 131.1, 131.6, 133.2, 135.6, 157, 165.2; HRMS (ESI) calc
for [M+H].sup.+ C.sub.18H.sub.20N.sub.2O.sub.4SBr 439.0327, obsd
439.0349; HPLC purity 94.34%, R.sub.T 13.35.
Example 2
1-[(2-bromo-5-methoxybenzoyl)]-4-[(4-fluorophenyl)sulfonyl]piperazine
[0168] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-fluorophenylsulfonylchloride (0.32 g, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 60:40) to give compound
(0.52 g, 68%). R.sub.f=0.33 (Cyclohexane/Ethyl acetate 60:40).
[0169] mp: 202.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.8-3.1 (m, 4H), 3.15-3.35 (m, 2H), 3.75 (m, 4H), 3.95 (m, 1H), 6.6
(d, J=3 Hz, 1H), 6.7 (dd, J=8.84 Hz, 3.02 Hz, 1H), 7.2 (m, 2H), 7.3
(d, J=8.85 Hz, 1H), 7.7 (m, 2H); .sup.13C NMR (75.46 MHz, CDCl3):
.delta. 40, 44.7, 45.1, 45.2, 54.7, 108.1, 112.2, 115.3, 115.7 (2C,
J=10 Hz), 129.5 (2C, J=9 Hz), 130.6 (J=130 Hz), 132.8, 136.8,
158.3, 162.8, 164.5 (CF, J=255 Hz); .sup.19F NMR (282.37 MHz,
CDCl3) 6-103.9 (m, 1F); HRMS (ESI) calc for [M+H.sup.+]
C.sub.18H.sub.19N.sub.2O.sub.4SBrF 457.0233, obsd 457.0213; HPLC
purity 100%, R.sub.T 13.76 min.
Example 3
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-trifluoromethylphenyl)sulfonyl]pipe-
razine
[0170] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-trifluoromethylphenylsulfonylchloride (0.42 g,
1.67 mmol) and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in
20 mL of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.6 g, 71%). R.sub.f=0.49 (Cyclohexane/Ethyl acetate 50:50).
[0171] mp: 155.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.90 (m, 1H), 3.05-3.15 (m, 3H), 3.2-3.4 (m, 2H), 3.7 (s, 3H,
OCH.sub.3), 3.75 (m, 1H), 3.9 (m, 1H) 6.65 (d, J=3 Hz, 1H), 6.7
(dd, J=8.8 Hz, 3 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.7-7.9 (m, 4H);
.sup.13C NMR (75.46 MHz, CDCl3): .delta. 38.4, 43.1, 43.5, 43.6,
53.2, 106.5, 110.7, 114.2, 120.62 (q, CF.sub.3, J=273.2 Hz), 124
(2C, J=3.8 Hz), 125.7 (2C), 131.3, 132.4 (C--CF.sub.3, q, J=33 Hz),
135.1, 136.9, 156.7, 165; HRMS (ESI) calc for [M+H.sup.+]
C.sub.19H.sub.19N.sub.2O.sub.4SBrF.sub.3 507.0201, obsd 507.0184;
HPLC purity 100%, R.sub.T 15.29 min.
Example 4
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-acetylphenyl)sulfonyl]piperazine
[0172] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-acetylphenylsulfonylchloride (0.36 g, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.55 g, 68%). R.sub.f=0.15 (Cyclohexane/Ethyl acetate 50:50).
[0173] mp: 143.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.6 (s, 3H), 2.95 (m, 1H), 3-3.15 (m, 3H), 3.20-3.35 (m, 2H), 3.6
(s, 3H, OCH.sub.3), 3.7 (m, 1H), 3.9 (m, 1H), 6.6 (d, J=3 Hz, 1H),
6.7 (dd, J=8.8 Hz, 3.03 Hz, 1H), 7.3 (d, J=8.8 Hz, 1H), 7.8 (d,
J=6.8 Hz, 1.7 Hz, 2H), 8.05 (dd, J=6.8 Hz, 1.7 Hz, 2H); .sup.13C
NMR (75.46 MHz, CDCl3): .delta. 24.4, 38.4, 43.1, 43.5, 43.6, 53.2,
106.6, 110.7, 114.2, 125.5 (2C), 126.6 (2C), 131.3, 135.2, 137.1,
138, 156.7, 165, 194.1; HRMS (ESI) calc for [M+H.sup.+]
C.sub.20H.sub.22N.sub.2O.sub.5SBr 481.0433, obsd 481.0437; HPLC
purity 95.5%, R.sub.T 13.17 min.
Example 5
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-propylphenyl)sulfonyl]piperazine
[0174] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-propylphenylsulfonylchloride (0.3 mL, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Petroleum ether/Ethyl acetate 50:50) to give
compound (0.62 g, 77%). R.sub.f=0.45 (Petroleum ether/Ethyl acetate
50:50).
[0175] mp: 134.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
0.88 (t, J=7.3 Hz, 3H), 1.61 (m, 2H), 2.61 (t, J=7.6 Hz, 2H),
2.86-3.1 (m, 4H), 3.17-3.36 (m, 2H), 3.67-3.75 (m, 1H), 3.69 (s,
3H, OCH.sub.3), 3.87-3.95 (m, 1H), 6.6 (d, J=3 Hz, 1H), 6.7 (dd,
J=8.8 Hz, 3 Hz, 1H), 7.25-7.35 (m, 3H), 7.65 (d, J=8.35 Hz, 2H);
.sup.13C NMR (75.46 MHz, CDCl3): .delta. 15.3, 25.8, 39.5, 42.6,
47.3, 47.7, 47.8, 57.3, 110.7, 114.8, 118.3, 129.4 (2C), 130.9
(2C), 134.1, 135.4, 139.4, 150.4, 160.8, 169.1; HRMS (ESI) calc for
[M+H].sup.+ C.sub.21H.sub.26N.sub.2O.sub.4SBr 481.0797, obsd
481.0777; HPLC purity 100%, R.sub.T 15.95 min.
Example 6
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-nitrophenyl)sulfonyl]piperazine
[0176] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-nitrophenylsulfonylchloride (0.37 g, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Petroleum ether/Ethyl acetate 50:50) to give
compound (0.62 g, 77%). R.sub.f=0.37 (Petroleum ether/Ethyl acetate
50:50).
[0177] mp: 151.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.9-3.35 (m, 6H), 3.65 (s, 3H, OCH.sub.3), 3.7 (m, 1H), 3.85 (m,
1H), 6.6 (d, J=3 Hz, 1H), 6.75 (dd, J=8.9 Hz, 3 Hz, 1H), 7.35 (d,
J=8.8 Hz, 1H), 7.85 (d, J=2.3 Hz, 2H), 8.35 (d, J=2.3 Hz, 2H);
.sup.13C NMR (75.46 MHz, CDCl3): .delta. 40.9, 45.6, 46, 46.1,
55.7, 109, 113.2, 116.7, 124.5 (2C), 128.8 (2C), 133.8, 137.5,
141.8, 150.4, 159.2, 167.4; HRMS (ESI) calc for [M+H].sup.+
C.sub.18H.sub.19N.sub.3O.sub.6SBr 484.0178, obsd 484.0186; HPLC
purity 97%, R.sub.T 14 min.
Example 7
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-trifluoromethylphenyl)sulfonyl]pipe-
razine
[0178] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 3-trifluoromethylphenylsulfonylchloride (0.28 mL,
1.67 mmol) and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in
20 mL of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.69 g, 81%). R.sub.f=0.44 (Cyclohexane/Ethyl acetate 50:50).
[0179] mp: 154.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.90 (m, 1H), 3.05-3.15 (m, 3H), 3.2-3.4 (m, 2H), 3.7 (s, 3H,
OCH.sub.3), 3.75 (m, 1H), 3.9 (m, 1H) 6.65 (d, J=3 Hz, 1H), 6.7
(dd, J=8.8 Hz, 3 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.65 (t, J=7.8
Hz, 1H), 7.8-7.95 (m, 3H); .sup.13C NMR (75.46 MHz, CDCl3): .delta.
40.9, 45.6, 46, 46.1, 55.6, 109, 113.1, 116.8, 123.1 (CF.sub.3,
J=273.2 Hz), 124.6 (J=3.8 Hz), 129.9 (J=3.8 Hz), 130.2, 130.8,
132.1 (C--CF.sub.3, J=34 Hz), 133.8, 137, 137.6, 159.2, 167.5; HRMS
(ESI) calc for [M+H.sup.+] C.sub.19H.sub.19N.sub.2O.sub.4SBrF.sub.3
507.0201, obsd 507.0182; HPLC purity 100%, R.sub.T 15.15 min.
Example 8
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-trifluoromethylphenyl)sulfonyl]pipe-
razine
[0180] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 2-trifluoromethylphenylsulfonylchloride (0.26 mL,
1.67 mmol) and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in
20 mL of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.51 g, 60%). R.sub.f=0.37 (Cyclohexane/Ethyl acetate 50:50).
[0181] mp: 136.5.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.15-3.45 (m, 6H), 3.75 (m, 4H), 4.05 (m, 1H), 6.75 (d, J=3
Hz, 1H), 6.8 (dd, J=8.8 Hz, 3 Hz, 1H), 7.45 (d, J=8.8 Hz, 1H), 7.7
(m, 2H), 7.85 (m, 1H), 8.05 (m, 1H); .sup.13C NMR (75.46 MHz,
CDCl3): .delta. 39.7, 43.7, 44, 45, 54.1, 107.6, 111.6, 115.3,
120.9 (CF.sub.3, J=274 Hz), 126.6 (C--CF.sub.3, J=33 Hz), 127.2
(CH, J=6.8 Hz), 130.6, 130.8, 131.6, 132.2, 135.7, 136.3, 157.7,
166; HRMS (ESI) calc for [M+H.sup.+]
C.sub.19H.sub.19N.sub.2O.sub.4SBrF.sub.3 507.0201, obsd 507.0185;
HPLC purity 98.5%, R.sub.T 14.67 min.
Example 9
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-biphenyl)sulfonyl]piperazine
[0182] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-biphenylsulfonylchloride (0.42 g, 1.67 mmol) and
N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
0.62 g, 72%). R.sub.f=0.47 (Cyclohexane/Ethyl acetate 50:50).
[0183] mp: 100.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.95 (m, 1H), 3-3.15 (m, 3H), 3.20-3.40 (m, 2H), 3.6 (s, 3H,
OCH.sub.3), 3.7 (m, 1H), 3.85 (m, 1H), 6.6 (d, J=3 Hz, 1H), 6.75
(dd, J=8.8 Hz, 3 Hz, 1H), 7.3-7.45 (m, 4H), 7.55 (d, 2H), 7.7 (m,
4H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 41, 45.7, 46.1, 46.2,
55.6, 109.1, 113.1, 116.7, 127.34, 127.8 (2C), 128.2 (2C), 128.7
(2C), 129.1 (2C), 133.7, 133.8, 137.7, 139, 146.2, 159.2, 167.5;
HRMS (ESI) calc for [M+H.sup.+] C.sub.24H.sub.24N.sub.2O.sub.4SBr
515.0640, obsd 515.0628; HPLC purity 97%, R.sub.T 16.05 min.
Example 10
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-methylphenyl)sulfonyl]piperazine
[0184] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and tosylchloride (0.32 g, 1.67 mmol) and
N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Petroleum ether/Ethyl acetate 50:50) to give
compound (0.51 g, 67%). R.sub.f=0.39 (Petroleum ether/Ethyl acetate
50:50).
[0185] mp: 178.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.35 (s, 3H), 2.85 (m, 1H), 3.00 (m, 3H), 3.15 (m, 1H), 3.25 (m,
1H), 3.65 (s, OCH.sub.3), 3.75 (m, 1H), 3.85 (m, 1H), 6.55 (d, J=3
Hz, 1H), 6.70 (dd, J=8.8 Hz, 3 Hz, 1H), 7.25-7.4 (m, 3H), 7.6 (d,
J=8.2 Hz, 2H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 19.6, 38.9,
43.7, 44.1, 44.1, 53.65, 107.1, 111.2, 114.6, 125.7 (2C), 127.9
(2C), 130.4, 131.7, 135.8, 142.2, 157.2, 165.4; HRMS (ESI) calc for
[M+H].sup.+ C.sub.19H.sub.22N.sub.2O.sub.4SBr 453.0483, obsd
453.0484; HPLC purity 99.6%, R.sub.T 14.16 min.
Example 11
[(2-bromo-5-methoxybenzoyl)]-4N-(4-trifluoromethoxyphenyl)sulfonyl]piperaz-
ine
[0186] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-trifluoromethoxyphenylsulfonylchloride (0.29 mL,
1.67 mmol) and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in
20 mL of CH.sub.2Cl.sub.2. The crude product was crystallized from
acetone to give pure product (0.6 g, 69%).
[0187] mp: 110.4.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 2.90 (m, 1H), 2.95-3.15 (m, 3H), 3.2-3.4 (m, 2H), 3.7 (s,
3H, OCH.sub.3), 3.75 (m, 1H), 3.9 (m, 1H) 6.65 (d, J=3 Hz, 1 H),
6.7 (dd, J=8.8 Hz, 3 Hz, 1H), 7.25-7.4 (m, 3H), 7.75 (m, 2H);
.sup.13C NMR (75.46 MHz, CDCl3): .delta. 40.9, 45.6, 46, 46.1,
55.6, 109, 113.1, 116.7, 118.47, 121.1 (2C), 129.8 (2C), 133.79,
133.9, 137.7, 152.6, 159.2, 167.5; .sup.19F NMR (282.37 MHz, CDCl3)
6-57.6 (s, 3F); HRMS (ESI) calc for [M+H.sup.+]
C.sub.19H.sub.19N.sub.2O.sub.5SBrF.sub.3 523.0150, obsd 523.0130;
HPLC purity 100%, R.sub.T 15.43 min.
Example 12
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2, 4
difluorophenyl)sulfonyl]piperazine
[0188] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 2,4-difluorophenylsulfonylchloride (0.22 mL, 1.67
mmol) and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL
of CH.sub.2Cl.sub.2. The crude product was crystallized from
acetone to give pure product (0.6 g, 75%).
[0189] mp: 194.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
3.15-3.45 (m, 6H), 3.75 (m, 4H), 4.05 (m, 1H), 6.75 (d, J=3 Hz,
1H), 6.8 (dd, J=8.8 Hz, 3 Hz, 1H), 7.05 (m, 2H), 7.45 (d, J=8.8 Hz,
1H), 7.90 (m, 1H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 41.2,
45.4, 45.8, 46.4, 55.6, 105.6 (J=25.7 Hz), 109.1, 112.2 (J=3.8 Hz),
113.2, 116.7, 121.7 (J=3.8 Hz), 132.9 (J=10.6 Hz), 133.8, 137.8,
159.2, 159.7 (J=261.1 Hz), 162.8 (J=258.8 Hz), 167.6; .sup.19F NMR
(282.4 MHz, CDCl3) 6-102.3 (m, 1F), -99.5 (m, 1F); HRMS (ESI) calc
for [M+H'] C.sub.18H.sub.18N.sub.2O.sub.4SBrF.sub.2475.0139, obsd
475.0143; HPLC purity 97.9%, R.sub.T 13.98 min.
Example 13
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-fluorophenyl)sulfonyl]piperazine
[0190] The general synthetic method described above affords as
white solid from 1-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 2-fluorophenylsulfonylchloride (0.22 mL, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.52 g, 68%).
[0191] mp: 167.3.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.10 (m, 1H), 3.15-3.4 (m, 5H), 3.7 (s, 3H, OCH.sub.3),
3.75 (m, 1H), 3.9 (m, 1H) 6.65 (d, J=3 Hz, 1H), 6.7 (dd, J=8.8 Hz,
3 Hz, 1H), 7.15-7.25 (m, 2H), 7.3 (d, J=8.8 Hz, 1H), 7.5 (m, 1H),
7.75 (m, 1H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 43, 47.1,
47.5, 48.2, 57.3, 110.8, 114.9, 118.4, 119.1 (J=21 Hz), 126.4 (J=3
Hz), 126.8 (J=14 Hz), 132.9, 135.5, 137.2 (J=8 Hz), 139.5, 160.6
(CF, J=255 Hz), 160.9, 169.2; HRMS (ESI) calc for [M+H.sup.+]
C.sub.18H.sub.19N.sub.2O.sub.4SbrF 457.0227, obsd 457.0233; HPLC
purity 100%, R.sub.T 13.57 min.
Example 14
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2,6-difluorophenyl)sulfonyl]piperazin-
e
[0192] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 2,6 difluorophenylsulfonylchloride (0.36 g, 1.67
mmol) and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL
of CH.sub.2Cl.sub.2. The crude product was crystallized from
acetone to give pure product (0.55 g, 69%).
[0193] mp: 154.5.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.15-3.45 (m, 6H), 3.75 (m, 4H), 3.95 (m, 1H) 6.65 (d, J=3
Hz, 1H), 6.7 (dd, J=8.8 Hz, 3 Hz, 1H), 7.05 (t, J=8.7 Hz, 2H), 7.45
(d, J=8.8 Hz, 1H), 7.5 (m, 1H); .sup.13C NMR (75.46 MHz, CDCl3):
.delta. 41.2, 45.3, 45.7, 46.4, 55.6, 109.1, 113.1, 113.5 (2C,
J=3.8 Hz), 115.2 (J=16.6 Hz), 116.7, 133.8, 135.1, 137.7, 159.2,
159.7 (J=259 Hz, 2C--F), 167.5; .sup.19F NMR (282.4 MHz, CDCl3)
.delta.-(m, 1F), -(m, 1F); HRMS (ESI) calc for [M+H.sup.+]
C.sub.18H.sub.18N.sub.2O.sub.4SBrF.sub.2 475.0139 obsd 475.0143;
HPLC purity 92.4%, R.sub.T 13.65 min.
Example 15
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-fluorophenyl)sulfonyl]piperazine
[0194] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 3-fluorophenylsulfonylchloride (0.23 mL, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.56 g, 73%).
[0195] mp: 145.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.93 (m, 1H), 3-3.15 (m, 3H), 3.20-3.40 (m, 2H), 3.7 (s, 3H,
OCH.sub.3), 3.75 (m, 1H), 3.9 (m, 1H) 6.6 (d, J=3 Hz, 1H), 6.7 (dd,
J=8.8 Hz, 3 Hz, 1H), 7.2-7.4 (m, 3H), 7.5 (m, 2H); .sup.13C NMR
(75.46 MHz, CDCl3): .delta. 40.9, 45.7, 46, 46.1, 55.6, 109.1,
113.2, 115 (J=24 Hz), 116.7, 120.5 (J=21 Hz), 123.4 (J=3.8 Hz),
131.1 (J=7.5 Hz), 133.8, 137.6 (J=6 Hz), 137.7, 159.2, 162.5 (J=252
Hz), 167.5; HRMS (ESI) calc for [M+H.sup.+]
C.sub.18H.sub.19N.sub.2O.sub.4SBrF 457.0233, obsd 457.0237; HPLC
purity 98.9%, R.sub.T 13.89 min.
Example 16
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-isopropylphenyl)sulfonyl]piperazine
[0196] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-isopropylphenylsulfonylchloride (0.3 mL, 1.67
mmol) and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL
of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Petroleum ether/Ethyl acetate 50:50) to give
compound (0.55 g, 68%). R.sub.f=0.52 (Petroleum ether/Ethyl acetate
50:50).
[0197] mp: 123.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
1.25 (s, 6H), 2.9-3.15 (m, 5H), 3.2-3.4 (m, 2H), 3.65 (s, 3H,
OCH.sub.3), 3.7 (m, 1H), 3.85 (m, 1H), 6.6 (d, J=3 Hz, 1H), 6.7
(dd, J=8.8 Hz, 3 Hz, 1H), 7.4 (m, 3H), 7.65 (d, J=8.4 Hz, 2H);
.sup.13C NMR (75.46 MHz, CDCl3): .delta. 23.6 (2 C), 34.2, 41,
45.6, 46.1, 46.1, 55.6, 109.1, 113.1, 116.6, 127.3 (2C), 127.9
(2C), 132.5, 133.7, 137.8, 154.8, 159.2, 167.4; HRMS (ESI) calc for
[M+H].sup.+ C.sub.21H.sub.26N.sub.2O.sub.4SBr 481.0797, obsd
481.0791; HPLC purity 99.6%, R.sub.T 15.77 min.
Example 17
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-bromophenyl)sulfonyl]piperazine
[0198] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-bromophenylsulfonylchloride (0.43 g, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.56 g, 65%).
[0199] mp: 180.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.9 (m, 1H), 3.05 (m, 3H), 3.15-3.35 (m, 2H), 3.65 (s, 3H,
OCH.sub.3), 3.7 (m, 1H), 3.85 (m, 1H), 6.6 (d, J=3 Hz, 1H), 6.75
(dd, J=8.9 Hz, 3 Hz, 1H), 7.3 (d, 1H), 7.5 (d, J=2.3 Hz, 2H), 7.65
(d, J=2.3 Hz, 2H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 40.9,
45.6, 46, 46.1, 55.7, 109.1, 113.2, 116.7, 128.4, 129.1 (2C), 132.6
(2C), 133.7, 134.6, 137.7, 159.2, 167.4; HRMS (ESI) calc for
[M+H].sup.+ C.sub.18H.sub.19N.sub.2O.sub.4SBr.sub.2 516.9432, obsd
516.9438; HPLC purity 98.2%, R.sub.T 14.93 min.
Example 18
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-chlorophenyl)sulfonyl]piperazine
[0200] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-chlorophenylsulfonylchloride (0.36 g, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.5 g, 63%).
[0201] mp: 188.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.9 (m, 1H), 2.95-3.05 (m, 3H), 3.15-3.35 (m, 2H), 3.65 (s, 3H,
OCH.sub.3), 3.7 (m, 1H), 3.85 (m, 1H), 6.6 (d, J=3 Hz, 1H), 6.7
(dd, J=8.8 Hz, 3 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.45 (d, J=Hz,
2H), 7.6 (d, J=Hz, 2H); .sup.13C NMR (75.46 MHz, CDCl3): .delta.
40.9, 45.6, 46, 46.1, 55.6, 109.1, 113.2, 116.7, 129.1 (2C), 129.6
(2C), 133.7, 134.1, 137.7, 139.9, 159.2, 167.4; HRMS (ESI) calc for
[M+H].sup.+ C.sub.18H.sub.19N.sub.2O.sub.4SBrC1472.9914, obsd
472.9937; HPLC purity 100%, R.sub.T 14.60 min.
Example 19
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(4-methoxyphenyl)sulfonyl]piperazine
[0202] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and 4-methoxyphenylsulfonylchloride (0.34 g, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.52 g, 66%). R.sub.f=0.296 (Cyclohexane/Ethyl acetate 50:50).
[0203] mp: 187.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.9 (m, 1H), 3.05 (m, 3H), 3.15-3.35 (m, 2H), 3.65 (s, OCH.sub.3),
3.7 (m, 1H), 3.75 (s, 3H, OCH.sub.3), 3.8 (m, 1H), 6.6 (d, J=3 Hz,
1H), 6.7 (dd, J=8.8 Hz, 3 Hz, 1H), 6.9 (m, 2H), 7.35 (d, J=8.8 Hz,
1H), 7.65 (m, 2H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 40.6,
45.4, 45.8, 45.8, 55.36, 55.40, 108.8, 112.9, 114.1 (2C), 116.4,
126.6, 129.6 (2C), 133.5, 137.5, 158.9, 163.1, 167.2; HRMS (ESI)
calc for [M+H.sup.+] C.sub.19H.sub.22N.sub.2O.sub.5SBr 469.0428,
obsd 469.0433; HPLC purity 100%, R.sub.T 13.66 min.
Example 20
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylmethane)sulfonyl]piperazine
[0204] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.5 g,
1.67 mmol), and phenylmethanesulfonylchloride (0.32 g, 1.67 mmol)
and N,N-diisopropyl ethyl amine (0.44 mL, 2.51 mmol) in 20 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 40:60) to give compound
(0.5 g, 66%). R.sub.f=0.32 (Cyclohexane/Ethyl acetate 50:50).
[0205] mp: 112.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
2.95 (m, 4H), 3.1-3.3 (m, 2H), 3.45 (m, 1H), 3.65 (s, 3H,
OCH.sub.3), 3.7 (s, 3H), 3.9 (m, 1H), 4.2 (s, 2H), 6.6 (d, J=3 Hz,
1H), 6.7 (dd, J=8.8 Hz, 3 Hz, 1H), 7.3 (m, 6H); .sup.13C NMR (75.46
MHz, CDCl3): .delta. 41.9, 45.9, 46.4, 47.2, 55.9, 57.8, 109.3,
113.3, 117, 128.6, 129.2 (2C), 129.3, 130.9 (2C), 134, 138.1,
159.5, 167.7; HRMS (ESI) calc for [M+H.sup.+]
C.sub.19H.sub.22N.sub.2O.sub.4SBr 453.0484, obsd 453.0483; HPLC
purity 99%, R.sub.T 13.54 min.
Example 21
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-thiophene)sulfonyl]piperazine
[0206] The general synthetic method described above affords as
white solid from 1N-(2-bromo-5-methoxybenzoyl)piperazine (0.25 g,
0.84 mmol), and 2-thiophenesulfonylchloride (0.158 g, 0.84 mmol)
and N,N-diisopropyl ethyl amine (0.22 mL, 1.25 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.27 g, 72%).
[0207] mp: 155.8.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 2.95 (m, 1H), 3-3.2 (m, 4H), 3.2-3.4 (m, 1H), 3.75 (m, 4H),
3.95 (m, 1H), 6.65 (d, J=3 Hz, 1H), 6.7 (dd, J=8.8 Hz, 3 Hz, 1H),
7.05 (t, J=Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.45 (dd, J=3.7 Hz, 1.3
Hz, 1H), 7.6 (dd, J=5 Hz, 1.3 Hz, 1H); HRMS (ESI) calc for
[M+H.sup.+] C.sub.16H.sub.18N.sub.2O.sub.4S.sub.2Br 441.9891, obsd
441.9883; HPLC purity 99.5%, R.sub.T 13.32 min.
Example 22
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-thiophene)sulfonyl]piperazine
[0208] The general synthetic method described above affords as
white solid from 1-(2-bromo-5-methoxybenzoyl)piperazine (0.25 g,
0.84 mmol), and 3 thiophenesulfonylchloride (0.158 g, 0.84 mmol)
and N,N-diisopropyl ethyl amine (0.22 mL, 1.25 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.27 g, 67%).
[0209] mp: 156.9.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 2.90-3.35 (m, 6H), 3.75 (m, 4H), 3.95 (m, 1H), 6.60 (d, J=3
Hz, 1H), 6.7 (dd, J=8.8 Hz, 3 Hz, 1H), 7.2 (m, 1H), 7.35 (d, J=8.8
Hz, 1H), 7.45 (dd, J=5.1 Hz, 3.1 Hz, 1H), 7.85 (dd, J=3 Hz, 1.3 Hz,
1H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 38.50, 43.19, 43.59,
43.69, 53.24, 106.67, 110.72, 114.29, 123.36, 125.81, 128.81,
131.36, 133.15, 135.34, 156.81, 165.05; HRMS (ESI) calc for
[M+H.sup.+] C.sub.16H.sub.18N.sub.2O.sub.4S.sub.2Br 441.9891, obsd
441.9884; HPLC purity 100%, R.sub.T 12.92 min.
Example 23
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(2-furan)sulfonyl]piperazine
[0210] The general synthetic method described above affords as
white solid from 1-(2-bromo-5-methoxybenzoyl)piperazine (0.25 g,
0.84 mmol), and 2-furanesulfonylchloride (0.140 g, 0.84 mmol) and
N,N-diisopropyl ethyl amine (0.22 mL, 1.25 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.23 g, 64%).
[0211] mp: 132.8.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.05-3.35 (m, 6H), 3.75 (m, 4H), 3.95 (m, 1H), 6.45 (dd,
J=3.5 Hz, 1.8 Hz, 1H), 6.65 (d, J=3 Hz, 1H), 6.75 (dd, J=8.8 Hz, 3
Hz, 1H), 7 (dd, J=3.5 Hz, 0.8 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.5
(dd, J=1.7 Hz, 0.8 Hz, 1H); .sup.13C NMR (75.46 MHz, CDCl3):
.delta. 40.95, 45.54, 45.94, 46.14, 55.66, 109.09, 111.38, 113.18,
116.70, 117.55, 133.79, 137.76, 146.45, 146.85, 159.23, 167.51;
HRMS (ESI) calc for [M+H.sup.+] C.sub.16H.sub.18N.sub.2O.sub.5SBr
429.0120, obsd 429.0122; HPLC purity 100%, R.sub.T 12.87 min.
Example 24
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(3-furan)sulfonyl]piperazine
[0212] The general synthetic method described above affords as
white solid from 1-(2-bromo-5-methoxybenzoyl)piperazine (0.25 g,
0.84 mmol), and 3-furanesulfonylchloride (0.140 g, 0.84 mmol) and
N,N-diisopropyl ethyl amine (0.22 mL, 1.25 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was crystallized from acetone
to give pure product (0.26 g, 72%).
[0213] mp: 162.3.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 2.9-3.4 (m, 6H), 3.75 (m, 4H), 3.95 (m, 1H), 6.45 (dd,
J=1.9 Hz, 0.7 Hz, 1H), 6.65 (d, J=3 Hz, 1H), 6.75 (dd, J=8.8 Hz, 3
Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.45 (t, J=1.8 Hz, 1H), 7.85 (dd,
J=1.4 Hz, 0.7 Hz, 1H); .sup.13C NMR (75.46 MHz, CDCl3): .delta.
40.85, 45.51, 45.89, 46.05, 55.66, 108.6, 109.08, 113.17, 116.72,
122.74, 133.78, 137.75, 144.97, 146.06, 159.24, 167.47; HRMS (ESI)
calc for [M+H].sup.+ C.sub.16H.sub.18N.sub.2O.sub.5SBr 429.0120,
obsd 429.0110; HPLC purity 100%, R.sub.T 12.46 min.
Example 25
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(1-methyl-1H-imidazole)sulfonyl]pipera-
zine
[0214] The general synthetic method described above affords as
white solid from 1-(2-bromo-5-methoxybenzoyl)piperazine (0.25 g,
0.84 mmol), and 1-methyl-1H-imidazolesulfonylchloride (0.151 g,
0.84 mmol) and N,N-diisopropyl ethyl amine (0.22 mL, 1.25 mmol) in
10 mL of CH.sub.2Cl.sub.2. The crude product was crystallized from
acetone to give pure product (0.29 g, 78%).
[0215] mp: 190.2.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.1-3.4 (m, 6H), 3.75 (s, 6H), 3.95 (m, 2H), 6.65 (d, J=3
Hz, 1H), 6.75 (dd, J=8.8 Hz, 3 Hz, 1H), 7.35 (s, 1H), 7.38 (m, 1H),
7.43 (m, 1H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 34.08,
40.95, 45.75, 46.25, 46.32, 55.65, 109.14, 113.20, 116.60, 124.80,
133.72, 137.68, 137.93, 139.36, 159.17, 167.49; HRMS (ESI) calc for
[M+H.sup.+] C.sub.16H.sub.20N.sub.4O.sub.4SBr 443.0402, obsd
443.0389; HPLC purity.
Example 26
1N-[(3-bromo-4-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne
[0216] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.27 g,
1.03 mmol), and 3-bromo-4-methoxybenzoyl chloride (0.26 g, 1.03
mmol) and N,N-diisopropyl ethyl amine (0.27 mL, 1.56 mmol) in 10 mL
of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.31 g, 63%). R.sub.f=0.33 (Cyclohexane/Ethyl acetate 50:50).
[0217] mp: 166.7.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.15 (m, 4H), 3.75 (m, 4H), 3.85 (s, 3H), 6.7 (d, J=8.8 Hz,
1H), 6.9 (m, 2H), 7.25 (d, 2.5 Hz, 1H), 7.4 (dd, J=8.8 Hz, 2.5 Hz,
1H), 7.8 (m, 1H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 45.73,
56.40, 106.01 (J=25.7 Hz), 111.47, 111.84, 112.25 (J=3.8 Hz), 121.4
(J=3.8 Hz), 127.99, 128.15, 132.91 (J=9.8 Hz), 157.47, 159.78
(J=258.8 Hz), 166 (J=258.8 Hz), 168.98; HRMS (ESI) calc for
[M+H.sup.+] C.sub.18H.sub.18N.sub.2O.sub.4SBrF.sub.2 475.0139, obsd
475.0152; HPLC purity 97.7%, R.sub.T 14.11 min.
Example 27
1N-[(2-methoxy-5-bromobenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne
[0218] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.99 mmol), and 2-methoxy-5-bromobenzoyl chloride (0.25 g, 0.99
mmol) and N,N-diisopropyl ethyl amine (0.26 mL, 1.49 mmol) in 10 mL
of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.32 g, 68%). R.sub.f=0.49 (Cyclohexane/Ethyl acetate 50:50).
[0219] mp: 171.2.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 2.95-3.4 (m, 6H), 3.70 (s, 3H), 3.85 (m, 2H), 6.8 (d, J=8.5
Hz, 1H), 6.9 (m, 2H), 7.25 (dd, J=8.5 Hz, 2.1 Hz, 1H), 7.55 (d,
J=2.1 Hz, 1H), 7.8 (m, 1H); .sup.13C NMR (75.46 MHz, CDCl3):
.delta. 41.13, 45.34, 45.78, 46.32, 55.68, 105.77 (J=25.7 Hz),
112.05 (J=3.8 Hz), 112.61, 113.21, 121.45 (J=3.8 Hz), 126.49,
130.77, 132.89 (J=10.6 Hz), 133.43, 154.14, 158.1 (J=258.8 Hz),
166.01, 167.1 (J=258.8 Hz); HRMS (ESI) calc for [M+H.sup.+]
C.sub.18H.sub.18N.sub.2O.sub.4SBrF.sub.2 475.0139, obsd 475.0134;
HPLC purity 97.5%, R.sub.T 14.24 min.
Example 28
1N-[(2-methoxy-3-bromobenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazi-
ne
[0220] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.99 mmol), and 2-methoxy-3-bromo benzoyl chloride (0.25 g, 0.99
mmol) and N,N-diisopropyl ethyl amine (0.26 mL, 1.49 mmol) in 10 mL
of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.36 g, 76%). R.sub.f=0.51 (Cyclohexane/Ethyl acetate 50:50).
[0221] mp: 152.3.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.05-3.4 (m, 6H), 3.70 (3, 4H), 3.95 (m, 1H), 6.9 (m, 3H),
7.1 (dd, J=7.6 Hz, 1.6 Hz, 1H), 7.55 (dd, J=7.9 Hz, J=1.6 Hz, 1H),
7.8 (m, 1H); .sup.13C NMR (75.46 MHz, CDCl3): .delta. 41.48, 45.53,
45.94, 46.76, 62.10, 105.9 (J=25.7 Hz), 112.2 (J=3.8 Hz), 117.63,
121.74 (J=3.8 Hz), 125.98, 127.27, 131.44, 132.47 (J=12.8 Hz),
134.87, 152.99, 159.75 (J=258.8 Hz), 165.95 (J=258.8 Hz), 166.78;
HRMS (ESI) calc for [M+H.sup.+]
C.sub.18H.sub.18N.sub.2O.sub.4SBrF.sub.2 475.0139, obsd 475.0137;
HPLC purity 98.3%, R.sub.T 14.29 min.
Example 29
1N-[(4-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0222] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.99 mmol), and 4-methoxybenzoyl chloride (0.17 g, 0.99 mmol) and
N,N-diisopropyl ethyl amine (0.26 mL, 1.49 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.2 g, 51%). R.sub.f=0.35 (Cyclohexane/Ethyl acetate 50:50).
[0223] mp: 143.3.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.15 (m, 4H), 3.65 (m, 4H), 3.75 (s, 3H), 6.81-6.86 (m,
2H), 6.88-6.99 (m, 2H), 7.26-7.29 (m, 2H), 7.76-7.83 (m, 1H);
.sup.13C NMR (75.46 MHz, CDCl3): .delta. 45.79, 55.38, 106.05
(J=25.7 Hz), 112.2 (J=3.8 Hz), 113.88 (2C), 121.44 (J=3.8 Hz),
126.72, 129.30 (2C), 133.02 (J=2.3 Hz), 158.06 (J=258.8 Hz),
161.19, 165.96 (J=258.8 Hz), 166.78; HRMS (ESI) calc for
[M+H.sup.+] C.sub.18H.sub.19N.sub.2O.sub.4SF.sub.2 397.1034, obsd
397.1042; HPLC purity 97.3%, R.sub.T 12.98 min.
Example 30
1N-[(4-bromobenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0224] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.99 mmol), and 4-bromo benzoyl chloride (0.25 g, 0.99 mmol) and
N,N-diisopropyl ethyl amine (0.26 mL, 1.49 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.24 g, 54%). R.sub.f=0.66 (Cyclohexane/Ethyl acetate 50:50).
[0225] mp: 164.5.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.15 (m, 4H), 3.65 (br, 4H), 6.89-7 (m, 2H), 7.17 (m, 2H),
7.48 (m, 2H), 7.76-7.86 (m, 1H); HRMS (ESI) calc for [M+H.sup.+]
C.sub.17H.sub.16N.sub.2O.sub.3SBrF.sub.2 445.0033, obsd 445.0057;
HPLC purity 96.56%, R.sub.T 14.29 min.
Example 31
1N-[(3-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0226] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.99 mmol), and 3-methoxybenzoyl chloride (0.17 g, 0.99 mmol) and
N,N-diisopropyl ethyl amine (0.26 mL, 1.49 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.22 g, 56%). R.sub.f=0.42 (Cyclohexane/Ethyl acetate 50:50).
[0227] .sup.1H NMR (300.13 MHz, CDCl3): .delta. 3.3 (br, 4H),
3.7-4.9 (m, 7H), 7 (m, 2H), 7.05-7.17 (m, 3H), 7.41 (m, 1H), 7.96
(m, 1H). .sup.13C NMR (75.46 MHz, CDCl3): .delta. 45.78 (4C),
55.39, 106 (J=25.7 Hz), 112.3 (J=3.8 Hz), 112.69, 115.84, 119.03,
121.53 (J=3.8 Hz), 129.78, 133.02 (J=2.3 Hz), 136.07, 158.2
(J=258.8 Hz), 159.75, 165.82 (J=258.8 Hz), 170.3; HRMS (ESI) calc
for [M+H.sup.1] C.sub.18H.sub.19N.sub.2O.sub.4SF.sub.2 397.1034,
obs 397.1024.
Example 32
1N-[(2-methoxybenzoyl)]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0228] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.99 mmol), and 2-methoxybenzoyl chloride (0.17 g, 0.99 mmol) and
N,N-diisopropyl ethyl amine (0.26 mL, 1.49 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 30:70) to give compound
(0.2 g, 51%). R.sub.f=0.57 (Cyclohexane/Ethyl acetate 30:70).
[0229] mp: 140.8.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.05-3.5 (m, 6H), 3.75 (s, 3H), 3.9 (m, 2H), 6.9 (d, J=8.3
Hz, 1H), 6.93-7.05 (m, 3H), 7.2 (dd, J=7.5 Hz and 1.7 Hz, 1H), 7.3
(m, 1H), 7.85 (m, 1H). .sup.13C NMR (75.46 MHz, CDCl3): .delta.
41.17, 45.57, 45.99, 46.47, 55.45, 105.87 (J=25.7 Hz), 110.94,
112.16 (J=3.8 Hz), 121.13, 121.57 (J=3.8 Hz), 124.76, 128.13,
130.92, 133.05 (J=2.3 Hz), 155.15, 159.75 (J=258.8 Hz), 165.9
(J=258.8 Hz), 167.89; HRMS (ESI) calc for [M+H.sup.+]
C.sub.18H.sub.19N.sub.2O.sub.4SF.sub.2 397.1034, obsd 397.1022;
HPLC purity 96.99%, R.sub.T 12.83 min.
Example 33
1N-[isonicotinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0230] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.98 mmol), and isonicotinoyl chloride (0.14 g, 0.98 mmol) and
N,N-diisopropyl ethyl amine (0.25 mL, 1.49 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Acetone 30:70) to give compound (0.2 g,
56%). R.sub.f=0.51 (Cyclohexane/Acetone 30:70).
[0231] mp: 130.1.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.2 (m, 4H), 3.6 (m, 4H), 6.9 (m, 2H), 7.3 (m, 1H), 7.6 (m,
1H), 7.8 (m, 1H), 8.5 (m, 2H); HRMS (ESI) calc for [M+H.sup.+]
C.sub.16H.sub.16N.sub.3O.sub.3SF.sub.2 368.0880, obsd 368.0886;
HPLC purity 81.02%, R.sub.T 9.54 min.
Example 34
1N-[nicotinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0232] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.98 mmol), and nicotinoyl chloride (0.14 g, 0.98 mmol) and
N,N-diisopropyl ethyl amine (0.25 mL, 1.49 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Acetone 30:70) to give compound (0.19
g, 53%). R.sub.f=0.40 (Cyclohexane/Acetone 30:70).
[0233] mp: 148.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
3.10 (m, 4H), 3.4 (m, 2H), 3.8 (m, 2H), 6.8 (m, 2H), 7.1 (m, 2H),
7.8 (m, 1H), 8.6 (dd, J=4.35 Hz and J=1.6 Hz); HRMS (ESI) calc for
[M+H.sup.+] C.sub.16H.sub.16N.sub.3O.sub.3SF.sub.2 368.0880, obsd
368.0881; HPLC purity 86.6%, R.sub.T 9.37 min.
Example 35
1N-[picolinoyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0234] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.26 g,
0.98 mmol), and picolinoyl chloride (0.14 g, 0.98 mmol) and
N,N-diisopropyl ethyl amine (0.25 mL, 1.49 mmol) in 10 mL of
CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Acetone 30:70) to give compound (0.2 g,
56%). R.sub.f=0.76 (Cyclohexane/Acetone 30:70).
[0235] mp: 173.6.degree. C. .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.2 (m, 4H), 3.7 (m, 2H), 3.8 (m, 2H), 6.9 (m, 2H), 7.25
(m, 1H), 7.6 (dd, J=, 1H), 7.8 (m, 2H), 8.5 (m, 1H); .sup.13C NMR
(75.46 MHz, CDCl3): .delta. 42.07, 45.56, 46.14, 46.86, 105.99
(J=25.7 Hz), 112.23 (J=3.8 Hz), 122.2 (J=3.8 Hz), 124.48, 124.99,
133.04 (J=2.3 Hz), 137.30, 148.17, 153.13, 159.77 (J=258.8 Hz),
165.43 (J=258.8 Hz), 167.42; HRMS (ESI) calc for [M+H.sup.+]
C.sub.16H.sub.16N.sub.3O.sub.3SF.sub.2 368.0879, obsd 368.0881;
HPLC purity 94.23% R.sub.T 13.12 min.
Example 36
1N-[3-thiophenecarbonyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0236] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.25 g,
0.94 mmol), and 3-thiophenecarboxylic acyl chloride (0.14 g, 0.94
mmol) and N,N-diisopropyl ethyl amine (0.25 mL, 1.41 mmol) in 10 mL
of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.175 mg, 50%). R.sub.f=0.43 (Cyclohexane/Ethyl acetate
50:50).
[0237] mp: 157.3.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3):
.delta. 3.2 (m, 4H), 3.8 (m, 4H), 7 (m, 2H), 7.15 (dd, J=1.26 and 5
Hz, 1H), 7.35 (dd, J=2.95 and 5 Hz, 1H), 7.5 (dd, J=1.26 and 2.95
Hz, 1H), 7.95 (m, 1H). .sup.13C NMR (75.46 MHz, CDCl3): .delta.
45.58 (4C), 105.76 (J=25.7 Hz), 111.99 (J=3.8 Hz), 121.2 (J=3.8
Hz), 126.22, 126.62, 126.97, 132.81 (J=2.3 Hz), 135.22, 157.8
(J=258.8 Hz), 165.52 (J=258.8 Hz), 165.62; HRMS (ESI) calc for
[M+H.sup.+] C.sub.15H.sub.15N.sub.2O.sub.3S.sub.2F.sub.2 373.0492,
obsd 373.0484; HPLC purity 100%, R.sub.T 12.26 min.
Example 37
1N-[4-thiazolecarbonyl]-4-N-[(2,4-difluorophenyl)sulfonyl]piperazine
[0238] The general synthetic method described above affords as
white solid from 4N-(2,4-difluorophenylsulfonyl)piperazine (0.24 g,
0.93 mmol), and 4-thiazole carboxylic acyl chloride (0.14 g, 0.93
mmol) and N,N-diisopropyl ethyl amine (0.24 mL, 1.39 mmol) in 10 mL
of CH.sub.2Cl.sub.2. The crude product was purified by column
chromatography (Cyclohexane/Ethyl acetate 50:50) to give compound
(0.22 g, 63%). R.sub.f=0.17 (Cyclohexane/Ethyl acetate 50:50).
[0239] mp: 196.degree. C.; .sup.1H NMR (300.13 MHz, CDCl3): .delta.
3.2 (m, 4H), 4 (m, 4H), 6.8 (m, 2H), 7.8 (m, 1H), 8 (d, J=2.2 Hz,
1H), 8.7 (d, J=2.2 Hz, 1H). .sup.13C NMR (75.46 MHz, CDCl3):
.delta.42.21, 45.62, 46.26, 46.73, 105.96 (J=25.7 Hz), 112.17
(J=3.8 Hz), 121.35 (J=3.8 Hz), 125.89, 133.05 (J=2.3 Hz), 150.92,
152.06, 159.81 (J=258.8 Hz), 162.31, 165.93 (J=258.8 Hz); HRMS
(ESI) calc for [M+H.sup.+]
C.sub.14H.sub.14N.sub.3O.sub.3S.sub.2F.sub.2 374.0445, obsd
374.0453; HPLC purity 99.19%, R.sub.T 10.82 min.
TABLE-US-00001 TABLE I (IIa) ##STR00019## mP example R.sub.1
R.sub.2 n .degree. C. 1 ##STR00020## ##STR00021## 0 180 2
##STR00022## ##STR00023## 0 202 3 ##STR00024## ##STR00025## 0 155 4
##STR00026## ##STR00027## 0 143 5 ##STR00028## ##STR00029## 0 134 6
##STR00030## ##STR00031## 0 151 7 ##STR00032## ##STR00033## 0 154 8
##STR00034## ##STR00035## 0 136.5 9 ##STR00036## ##STR00037## 0 100
10 ##STR00038## ##STR00039## 0 178 11 ##STR00040## ##STR00041## 0
110.4 12 ##STR00042## ##STR00043## 0 194 13 ##STR00044##
##STR00045## 0 167.3 14 ##STR00046## ##STR00047## 0 154.5 15
##STR00048## ##STR00049## 0 145 16 ##STR00050## ##STR00051## 0 123
17 ##STR00052## ##STR00053## 0 180 18 ##STR00054## ##STR00055## 0
188 19 ##STR00056## ##STR00057## 0 187 20 ##STR00058## ##STR00059##
1 112 21 ##STR00060## ##STR00061## 0 155.8 22 ##STR00062##
##STR00063## 0 156.9 23 ##STR00064## ##STR00065## 0 132.8 24
##STR00066## ##STR00067## 0 162.3 25 ##STR00068## ##STR00069## 0
190.2 26 ##STR00070## ##STR00071## 0 166.7 27 ##STR00072##
##STR00073## 0 171.2 28 ##STR00074## ##STR00075## 0 152.3 29
##STR00076## ##STR00077## 0 143.3 30 ##STR00078## ##STR00079## 0
164.5 31 ##STR00080## ##STR00081## 0 -- 32 ##STR00082##
##STR00083## 0 140.8 33 ##STR00084## ##STR00085## 0 130.1 34
##STR00086## ##STR00087## 0 148 35 ##STR00088## ##STR00089## 0
173.6 36 ##STR00090## ##STR00091## 0 157.3 37 ##STR00092##
##STR00093## 0 196
Example 38
Biological Experiments
[0240] The compound
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine (BZ36), and, depending on the biological assays,
compounds of the invention described in previous examples 1 to 37
have been subjected to biological assay(s) which demonstrate their
relevance as active substances in therapy and in particular in the
treatment of prostate cancer.
[0241] Material and Methods
[0242] Compound
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine (BZ36) was prepared according to the method described
in the patent US 2005/0119251 A1.
[0243] Human Prostate Tissue Samples
[0244] Human prostate tissue was collected from consenting
patients, after protocol approval by the local ethics committee
(CHU Henry Mondor, Creteil) and characterisation by urological
pathologist. Localized prostate cancer specimens from the
peripheral zone of the prostate were obtained from men who had
radical prostatectomy as treatment for their prostate cancer,
Gleason.gtoreq.7 (n=10). Non tumoral prostate specimens were
obtained from men with begnin hyperplasia of the prostate (HBP) who
had radical prostatectomy (n=10).
[0245] Cell Culture, Transient Transfections and RNA Interference
Experiments
[0246] The benign PNT2 prostate cell line, the androgen-sensitive
LNCaP and the androgen-independent C4-2 human prostate carcinoma
cell lines were purchased from American Type Culture Collection
(Manassas, Va.).
[0247] Monolayer cell cultures were maintained in a RPMI 1640 media
M 1-glutamine (Invitrogen, Cergy, France) supplemented with 10%
fetal calf serum (FCS), 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 10 mM HEPES and 1.0 mM sodium pyruvate (Invitrogen)
at 37.degree. C. in 5% CO.sub.2.
[0248] Primary MEFs were obtained from embryos at embryonic day
13.5 by standard methods.
[0249] Monolayer cell cultures were grown in Dulbecco's Modified
Eagle's Medium (DMEM) supplemented with 25 mM glucose and 10%
FCS.
[0250] The transcriptional activity of the .beta.-catenin-Tcf4
complex was analysed by performing transient transfections with
0.25 .mu.g TCF/LEF-1 reporter (pTOP-FLASH) or control vector
(pFOP-FLASH).
[0251] Luciferase activities in cell lysates were normalized
relative to the .beta.-galactosidase activity to correct for
differences in transfection efficiency.
[0252] FIG. 3 shows representative results of at least two
independent experiments performed in triplicate.
[0253] For small interfering (si) RNA experiments, transfection of
LNCaP or C4-2 cells was carried out with pre-designed ON-TARGET
plus siRNA oligonucleotides control or targeting the human SCD-1
sequence GCACAUCAACUUCACCACA (Dharmacon, Lafayette, Co, USA).
[0254] Nucleofaction of cells with 2.5 .mu.g siRNA were performed
on 2.times.10.sup.6 cells using Amaxa nucleofactor R kit (Lonza,
Cologne, Germany). Twenty-four hours and 48 hours after
transfection, cells were processed for cell proliferation by BrdU
staining and harvested for RNA and protein analysis.
[0255] Animal Experiments
[0256] Male athymic nude Mice (Foxn1 nu/nu) (Harlan, Grannat,
France) were used at the age of 7 weeks (weight 25-30 g). All
procedures were performed in compliance with the European
Convention for the Protection of Vertebrate Animals Used for
Experimentation (animal house agreement # B-34-172-27,
authorization for animal experimentation #34.324). Experiments were
done at least twice for each tested condition. Animals were
sacrificed before they became compromised. Xenografts were
established by subcutaneously injecting 2.times.10.sup.6 LNCaP
cells, 2.times.10.sup.6 C4-2 cells or 5.times.10.sup.5 MEFs SV40 in
100 .mu.l of a Matrigel solution. For curative experiments, tumors
were allowed to growth until they were measurable with a caliper.
In each group, the mice were randomized and given SCD-1 inhibitor
at 80 mg/kg in 100 .mu.l of a labrafil-DMA-tween.sup.80 solution
(89:10:1) (treated group) or vehicle alone (control group), by
daily intra-peritoneal (i.p.) injection 5 days of week. For
preventive experiments, the mice were treated daily for 7 days
before the day of xenograft. Tumor volume measurements were taken
twice to three times a week and calculated according to the
formula: length.times.width.times.height.times.0.5236. Data are
expressed as the mean tumor volume or as fold of the start point
tumor volume. For survival analysis, animals bearing
pre-established C4-2 tumors were treated with BZ36 at 80 mg/kg or
160 mg/kg or with vehicle by daily i.p. injection 5 days of week.
All mice were monitored for survival until tumor volume had reached
2000 mm.sup.3 or until death. Analysis of survival was conducted by
a log-rank test based on the Kaplan-Meier method. At euthanasia,
tumors were excised and fixed in 4% formalin for immunohistological
analyses.
[0257] Rna Isolation, Reverse Transcription and Quantitative
Real-Time PCR:
[0258] RNA was extracted with the use of TRI-Reagent (Euromedex,
Mundolsheim, France) according to the manufacturers'
recommendations. Reverse transcription of total RNA was performed
at 37.degree. C. using the M-MLV reverse transcriptase (Invitrogen)
and random hexanucleotide primers (Promega, Madison, Wis.),
followed by a 15 min inactivation at 70.degree. C. Quantitative PCR
was conducted using the primers specific for human SCD1 and SYBR
Green Light Cycler Master Mix (Eurofins MWG Operon, Roissy CDG).
Measurement and analysis of gene expression were performed using
the ABI Prism 7300 Sequence detection System software (Applied
Biosystems), under the following conditions: 2 minutes at
50.degree. C. and 10 minutes at 95.degree. C.; and then 40 cycles
of 15 seconds at 95.degree. C. and 1 minute at 60.degree. C. The
relative content of cDNA samples was normalized by substracting the
threshold cycle (C.sub.t) of the endogenous 18S reference gene to
the target gene (.DELTA.C.sub.t.dbd.C.sub.t of target gene -C.sub.t
of 18S). Values are expressed as the relative mRNA level of
specific gene expression as obtained using the formula
2.sup.-(.DELTA.Ct).
[0259] C4-2 cells treated with the compound tested at 25 .mu.M were
analysed for the expression profiles of 84 genes related to
Wnt-mediated signal transduction by using Human WNT Signaling
Pathway PCR Array according to the manufacturers' recommendations
(Tebu-Bio, Le Perray en Yvelines, France). The relative content of
cDNA samples was normalized with the endogenous .beta.2M, HPRT1,
RPL13 and GAPDH reference genes, and the relative mRNA level was
calculated according to the formula 2.sup.-(.DELTA.Ct). Values are
expressed as the fold change in relative mRNA level following
treatment of cells with the tested compound at 25 .mu.M, as
compared to control.
[0260] Proliferation Assay
[0261] LNCaP, C4-2, PNT2 or MEFs cells were seeded in triplicate
24-wells dishes at a density of 25.times.10.sup.4 cells/dish. At 24
h, 48 h, 72 h and 96 h following addition of increasing
concentrations of the tested compound in DMSO or DMSO alone, cells
were trypsinized, pelleted by centrifugation at 1200 rpm for 5 min,
resuspended in 500 .mu.l of culture medium and counted in an
hemocytometer.
[0262] MTT Assay
[0263] LNCaP, C4-2 or PNT2 cells were seeded in triplicate 24-wells
dishes at a density of 25.times.10.sup.4 cells/dish and cells
viability was tested after treatment with increasing concentrations
of the tested compound for 48 h. After 48 h, medium was removed and
250 .mu.l of a 5 mg/ml MTT (Sigma, St Louis, Mo., USA) solution in
PBS was added to each well. After 4 h incubation at 37.degree. C.,
the MTT solution was removed, 200 .mu.l of DMSO (Sigma) was added
and cells were incubated for 5 min. Two hundred microliters of each
samples was distributed in 96-well plates for an optical density
reading at 540 nm.
[0264] Flow Cytometry Analysis of Cell Cycle and Apoptosis
[0265] For cell cycle analysis, LNCaP, C4-2 or PNT2 cells were
plated at a density of 25.times.10.sup.5 cells/dish in 6-wells
dishes and treated with increasing concentrations of the tested
compound for 24 h or 48 h. Cells were then rinsed in PBS, pelleted
at 400 g for 5 min and maintained on ice for 20 min before
resuspension in a 25 propidium iodide (Sigma) solution. Cells were
kept overnight at 4.degree. C. and the percentages of cells in G1,
S and G2-M phases of the cell cycle were measured with a Coulter
Epics XL.TM. flow cytometer (Becton Dickinson) using 488-nm laser
excitation. For apoptosis experiment, MEFs were treated with the
tested compound at 25 .mu.M for 24 h, then rinced in PBS, pelleted
at 400 g for 5 min and stained with annexinV-FITC (Roche
Diagnostics, Meylan, France) for 15 min at 4.degree. C. The
percentage of annexin positive cells was immediately analysed using
488-nm laser excitation.
[0266] Protein Extracts and Immunoblot Analysis
[0267] Protein extracts and sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE), electrotransfer
and immunoblotting were performed as previously described (34). The
primary antibodies rabbit anti-AMPK, rabbit anti-phospho AMPK
(Thr172), rabbit anti-Akt and rabbit anti-phospho Akt were
purchased from Cell signalling Technology (Ozyme, Saint Quentin
Yvelines, France). The primary antibody mouse anti-tubuline.alpha.
was purchased from Lab Vision (Thermo Fisher Scientific, Microm
France, Francheville, France). The primary antibodies rabbit
anti-p44/p42 MAPK, rabbit anti-phospho p44/p42 MAPK
(Thr202/Tyr204), mouse anti-GSK3.alpha./.beta., and rabbit
anti-phospho GSK3.alpha./.beta. were kindly provided by Dr Gilles
Freiss. LNCaP and C4-2 cells treated with the tested compound at 25
.mu.M were analyzed for the relative phosphorylation of 46 kinase
phosphorylation sites using human phospho-kinase array kit
(Proteome Profiler.TM.) according to the manufacturers'
recommendations (R&D systems Europe, Lille, France).
[0268] Fatty Acid Analysis
[0269] Total prostate tissue lipid were extracted three times with
5 mL choloroform/methanol (2/1) and 500 .mu.L of water. Aliquots of
the lipid extracts were dried under gaseous nitrogen and were
dissolved in 100 .mu.L of a mixture and were separated by thin
layer chromatography in different lipid classes using a
hexane/ether/acetic acid (70/30/1; V/V) solvent system. To aid
visualization, standarts of triacylglycerol, of cholesteryl esters
and phospholipds were co-spotted with samples. Spots were
identified under UV light after spraying with
2',7'-dichlorofluorescein solution in ethanol and comparing with
authentic standarts. They were scrapped off the plates and were
converted to fatty methyl esters by transesterification with 3 mL
of methanol/H.sub.2SO.sub.4 (19/1; V/V) at 90.degree. C. for 30
min. The different solutions were neutralized with an 1 mL of
aqueous solution of 10% of K.sub.2CO.sub.3 and fatty methyl esters
were extracted with 5 mL of hexane. The fatty methyl esters were
dried under gaseous nitrogen and were subjected to gas
chromatography (GC) and identified by comparaison with standarts.
(Sigma Chemicals, St. Louis, Mo.). GC was conducted with a Thermo
GC fitted with a flame ionization detector. A supelcowax-10 fused
silica capillary column (60m.times.0.32 mm i.d, 0.25 .mu.M film
thickness) was used and oven temperature was programmed from
50.degree. C. to 200.degree. C., increased 20.degree. C. per
minute, held for 50 min, increased 10.degree. C. per min to
220.degree. C., and held for 30 min.
[0270] Determination of SCD Activity
[0271] Subconfluent LNCaP, C4-2, PNT2 of Ras SV40 MEFs cells grown
in 6-wells plate were incubated with the tested inhibitor at 25
.mu.M for 2 h in serum and fatty acid-free DMEM media supplemented
0.2% BSA. In this environment, the cells are solely dependent on
endogenous fatty acid synthesis for production of storage,
structural and signaling lipids. Trace amount of [.sup.14C]
palmitic acid were then added to the culture (0.5 .mu.Ci/well), and
cells were incubated for 6 more hours. At the end of the
incubation, total cell lipids were extracted and saponified, then
released fatty acids were esterified with boron trifluoride in
methanol for 90 min at 100.degree. C. The derived methyl esters
were separated by argentation TLC (Thermo Fisher Scientific)
following the procedure of Wilson and Sargen, using a solvent phase
consisting of hexane:ethyl ether (90:10, by vol) (35). Pure stearic
and oleic methyl ester acids were run in parallel to the samples.
Air-dried plates were scanned on a Phosphorlmager and fatty acid
spots on TLC were analysed with Phosphorlmager software. SCD
activity was expressed as the ratio of palmitoleic on palmitic
methyl ester acids and normalized to cellular DNA content.
[0272] Measurement of De Novo Fatty Acid Synthesis
[0273] The tested inhibitor was added overnight at a final
concentration of 25 .mu.M to subconfluent cultures of cells grown
in 6-wells plate in serum and fatty acid-free DMEM media
supplemented with 0.2% BSA. Cultures were then labeled in
triplicate with 1.0 .mu.Ci of [U--.sup.14C]-palmitate or -stearate
for 6 h, and total lipids were folch extracted with
chloroform/methanol. Labeled lipids were subjected to TLC in
hexane/diethyl ether/acetic acid 90:10:1 (V/V), to separate
cholesterol ester, triglycerides and phospholipids. Standards were
run for each of the lipid classes. After chromatography, labeled
lipid classes were quantified by scintillation counting and
radioactivity was normalized to DNA content.
[0274] PI(3,4,5)P.sub.3 Measurement
[0275] The production of PI(3,4,5)P.sub.3 (PIP3) in LNCaP and C4-2
prostate cancer cells was measured 24 h following exposure to
control medium or to medium supplemented with the inhibitor tested
at 25 .mu.M. The levels of produced PIP3 were quantified after
cellular lipids extraction using a PI(3,4,5)P.sub.3 mass ELISA kit
according to the manufacturer's instruction.
[0276] Immunofluorescence (IF) and Immunohistochemistry (1HC) of
hSCD-1
[0277] Immunohistochemical analysis of SCD-1 expression was
performed using high-density Tissue Microarray (TMA) slide (Accumax
array) with 39 prostate adenocarcinoma spots from different
patients (Gleason scores from 5 to 9) with corresponding normal
tissues. Immunohistochemical analysis of pcna expression was
performed on 5 .mu.M paraffin-embedded sections of LNCaP, C4-2 of
Ras SV40 MEFs tumor xenografts. Briefly, after antigen retrieval,
deparaffinized sections were blocked of Fc receptors with PBS
containing 5% goat serum and then incubated with corresponding
anti-SCD-1 mouse antibody, 1:50 (Abcam, Paris, France) or anti-pcna
mouse antibody, 1:50 (Santa Cruz) in PBS-Tween 0.1%, overnight at
4.degree. C. SCD-1 staining was revealed with a
peroxidase-conjugated anti-mouse secondary antibody, 1:100 (Jackson
Immunoresearch) and the DAB chromogen (DAKO, Glostrup, Denmark) as
substrate. Sections were counterstained with Mayer's hematoxylin.
Pcna staining was revealed by immunofluorescence using a
FITC-conjugated anti-mouse secondary antibody, 1:150 (Jackson
Immunoresearch). Sections were mounted in mowiol and analysed
rapidly.
[0278] Immunocytochemistry
[0279] Immunocytochemical analysis of SCD-1 expression was
performed on PNT2, LNCaP, and C4-2 cells grown on coverslip.
Briefly, after fixation in 4% PFA and permeabilization with 0.5%
Triton X-100, cells were incubated with blocking buffer (PBS-1%
BSA). SCD1 staining was detected with an anti-SCD-1 mouse primary
antibody, 1:50 (Abcam) for 1 hour at 37.degree. C. and revealed
with a FITC-conjugated anti-mouse secondary antibody (Jackson
Immunoresearch), 1:150 for 30 min at 37.degree. C. Slides were
mounted in mowiol and analysed rapidly.
[0280] BrDU Staining
[0281] LNCaP and C4-2 cells grown on coverslips were incubated for
4 hours with BrdU (100 .mu.M final) at 24 hours and 48 hours
following SCD-1 knock-down. Cells were then fixed and permeabilized
with cold methanol for 10 min at -20.degree. C. After 3 washes with
PBS, DNA was denaturated with 4NHCL for 10 min at RT, and cells
were incubated with blocking buffer (PBS-1% BSA). BrDU was then
detected with anti-BrDU monoclonal antibody 1:50 (Dako,
Carpinteria, Calif.) for 1 hour at 37.degree. C. After 3 washes
with PBS, cells were incubated with an FITC-conjugated anti-mouse
secondary antibody 1:150 (Jackson Immunoresearch) for 30 min at
37.degree. C., and slides were mounted in mowiol.
[0282] Statistical analysis were performed with unpaired Student's
t-test. Differences were considered statistically significant at
p<0.05. (* p<0.05; ** p<0.01 and *** p<0.001).
[0283] Results
[0284] MUFA Content and SCD-1 Expression are Increased During
Prostate Cancer Progression
[0285] As a first approach to fatty acid FA composition was
measured during prostate cancer progression. The ratios of total
MUFA to saturated fatty acids (SFA) was significantly increased in
cholesterol esters (CE, triacylglycerides (TAG) and phospholipids
(PL) lipid fractions in human prostate cancer tissue samples with
Gleason score .gtoreq.7 (FIG. 1A), suggesting the participation of
desaturase enzymes. Interestingly, among the analyzed MUFAs, those
produced by a .DELTA.9-desaturase activity, in majority
palmitoleate (16:1n-7) and oleate (18:1n-9), were the most abundant
in all lipid subclasses (data not shown). Furthermore, the specific
desaturation indexes 16:1n-7/16:0 (FIG. 1B) and 18:1n-9/18:0 (FIG.
1C) were increased in human prostate samples, supporting the
participation of the .DELTA.9-desaturase enzyme.
[0286] Consistent with this observation, stearoyl-CoA
.DELTA.9-desaturase 1 (SCD-1) was increased in cancer, compared to
normal human prostates both at mRNA (FIG. 1D) and protein levels
(FIG. 1E), as analyzed by QPCR and immunohistochemistry (1HC)
studies respectively.
[0287] SCD-1 expression was also increased, as measured by
immunofluorescence in the human prostate cancer cell lines LNCaP
and C4-2, compared to the non-tumoral PNT2 benign prostate cell
line (FIG. 1F).
[0288] Pharmacological and Genetic Inhibition of SCD-1 activity by
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine Induces Growth Arrest of LNCaP Cell Lines In Vitro
[0289] Compound
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine (BZ36) was tested.
[0290] The efficacy of the tested compound to inhibit SCD-1
activity has been evaluated by measuring the conversion of
exogenous [.sup.14C] saturated palmitic acid (16:0) to
monounsaturated palmitoleic acid (16:1n-7). Moreover, the effects
of inhibition of SCD-1 activity on lipid synthesis and
proliferation in prostate cancer cell lines were analysed.
[0291] In all three cell lines tested a marked reduction of the
labelled monounsaturated palmitoleic acid was observed in the cells
with BZ36 treated, compared to control cells (FIG. 2A-D).
[0292] Interestingly, basal SCD-1 activity was progressively
increased from non-tumoral PNT2 to androgen independent C4-2 cell
lines, further suggesting the implication of SCD-1 in prostate
cancer progression (FIG. 2D). Inhibition of SCD-1 activity with the
tested compound correlated with a dose dependent decrease in cell
proliferation of LNCaP, and C4-2 cancer cells, reaching 100%
inhibition at the maximal dose used (FIG. 2E-F).
[0293] Flow cytometry analysis further demonstrated the inhibitory
effects of the tested compoundin CaP cells, showing accumulation of
cells in the G0/G1 phase of the cell cycle, concomitant with a
decrease in the S phase (FIG. 2G-I).
[0294] Strikingly, no effect in proliferation was observed in the
non-cancerous PNT2 cell line, even at a maximal dose. Similar to
what observed using SCD1 small molecule inhibitors, genetic SCD-1
inhibition using siRNA technology blocked SCD1 mRNA (FIG. 3A) and
protein (FIG. 3B) expressions, and resulted in a marked decrease in
proliferation in both LNCaP (FIG. 3C), and C4-2 (FIG. 3D) cell
types. These results suggested that, first, SCD-1 activity and
lipid synthesis is required in prostate cancer cells in order to
proliferate. Second, that non-cancer cell do not require de novo
lipid synthesis, and therefore normal cells are not sensitive to
inhibition of this pathway. And third, that the inhibitory effects
of SCD-1 inhibitors are mediated by SCD-1 since the same effects
are observed when SCD-1 is depleted from the cells.
[0295] Proliferation of LNCaP and C4-2 cells in presence or absence
of 25 .mu.M of compounds prepared according to the examples 1 to 28
was measured by BrDu incorporation following 48 h of treatment with
the compounds Inhibition of SCD-1 activity with the indicated
tested compounds results in a decrease of the proliferation of C4-2
and LNCaP cells.
[0296] The results are disclosed in the following table II and
table III.
TABLE-US-00002 TABLE II (IIa) ##STR00094## % Inhibition of
proliferation at example R.sub.1 R.sub.2 n 25 .mu.M (BrDU) on C4-2
1 ##STR00095## ##STR00096## 0 22.7 .+-. 11.2 2 ##STR00097##
##STR00098## 0 37.2 .+-. 8.8 3 ##STR00099## ##STR00100## 0 65.8
.+-. 6.4 4 ##STR00101## ##STR00102## 0 36 .+-. 5.4 5 ##STR00103##
##STR00104## 0 26.8 .+-. 20.2 6 ##STR00105## ##STR00106## 0 0 7
##STR00107## ##STR00108## 0 31.6 .+-. 7.61 8 ##STR00109##
##STR00110## 0 84.4 .+-. 8.5 9 ##STR00111## ##STR00112## 0 60.5
.+-. 5.7 10 ##STR00113## ##STR00114## 0 0 11 ##STR00115##
##STR00116## 0 63 .+-. 4.1 12 ##STR00117## ##STR00118## 0 55.2 .+-.
0.5 13 ##STR00119## ##STR00120## 0 55.6 .+-. 3.1 14 ##STR00121##
##STR00122## 0 0 15 ##STR00123## ##STR00124## 0 35.7 .+-. 6 16
##STR00125## ##STR00126## 0 75.4 .+-. 10.8 17 ##STR00127##
##STR00128## 0 67.6 .+-. 8.2 18 ##STR00129## ##STR00130## 0 38.1
.+-. 7.1 19 ##STR00131## ##STR00132## 0 22.4 .+-. 7.7 20
##STR00133## ##STR00134## 1 46.3 .+-. 11.7 21 ##STR00135##
##STR00136## 0 58.3 .+-. 19.9 22 ##STR00137## ##STR00138## 0 28
.+-. 2.6 23 ##STR00139## ##STR00140## 0 48 .+-. 14 24 ##STR00141##
##STR00142## 0 28.4 .+-. 9.8 25 ##STR00143## ##STR00144## 0 0 26
##STR00145## ##STR00146## 0 68.2 .+-. 18.2 27 ##STR00147##
##STR00148## 0 26.2 .+-. 6.2 28 ##STR00149## ##STR00150## 55.4 .+-.
14 31 ##STR00151## ##STR00152## 0 25.3 .+-. 6.1 32 ##STR00153##
##STR00154## 0 0 36 ##STR00155## ##STR00156## 0 40 .+-. 7.6 37
##STR00157## ##STR00158## 0 37 .+-. 7.8
TABLE-US-00003 TABLE III (IIa) ##STR00159## % Inhibition of
proliferation at 25 .mu.M Examples R.sub.1 R.sub.2 n (BrDU) on
LNCaP Example 7 ##STR00160## ##STR00161## 0 16.7 Example 8
##STR00162## ##STR00163## 0 40.1 Example 9 ##STR00164##
##STR00165## 0 48.3 Example 10 ##STR00166## ##STR00167## 0 0
Example 11 ##STR00168## ##STR00169## 0 45.5 Example 12 ##STR00170##
##STR00171## 0 60.6 Example 13 ##STR00172## ##STR00173## 0 27.1
Example 15 ##STR00174## ##STR00175## 0 0 Example 18 ##STR00176##
##STR00177## 0 49.7 Example 20 ##STR00178## ##STR00179## 1 16.8
[0297] Accordingly, the inhibition of LNCap and C4-2 cells
proliferation is also observed with the tested compound.
[0298] Percentage of proliferative LNCaP and C4-2 cells in the S
phase of the cell cycle following 48 h of treatment in presence or
absence of 25 .mu.M of compounds prepared according to the examples
1 to 6 was measured. Inhibition of SCD-1 activity with the
indicated tested compounds decreased the percentage of
proliferative LNCaP and C.sub.4-2 cells in the S phase.
[0299] The so-obtained resultst are disclosed in the following
table IV.
TABLE-US-00004 TABLE IV (IIa) ##STR00180## % LNCaP cells % C4-2
cells in phase S in phase S example R.sub.1 R.sub.2 n at 25 .mu.M
at 25 .mu.M Cellules 20 22 non traitees 1 ##STR00181## ##STR00182##
0 18 20.1 2 ##STR00183## ##STR00184## 0 5 11.8 3 ##STR00185##
##STR00186## 0 10 2.8 4 ##STR00187## ##STR00188## 0 18 19.7 5
##STR00189## ##STR00190## 0 3 10.7 6 ##STR00191## ##STR00192## 0 15
19.8
[0300] Percentage of viable C4-2 cells as measured by MTT assay
following 48 h of treatment in the presence or absence of 25 .mu.M
of compounds prepared according to the examples 1 to 13, 15, 18 and
20 was evaluated. Inhibition of SCD-1 activity with the indicated
tested compounds decreased the cell viability.
[0301] The results are disclosed in the following table V and are
expressed as % of viable treated cells normalized to viable control
cells.
TABLE-US-00005 TABLE V (IIa) ##STR00193## Viable C4-2 cells at 25
.mu.M example R.sub.1 R.sub.2 n (% of control) Cellules non
traitees 1 ##STR00194## ##STR00195## 0 45 2 ##STR00196##
##STR00197## 0 49 6 ##STR00198## ##STR00199## 0 42 7 ##STR00200##
##STR00201## 0 44 8 ##STR00202## ##STR00203## 0 73 9 ##STR00204##
##STR00205## 0 90 10 ##STR00206## ##STR00207## 0 59 11 ##STR00208##
##STR00209## 0 74 12 ##STR00210## ##STR00211## 0 70 13 ##STR00212##
##STR00213## 0 56 15 ##STR00214## ##STR00215## 0 83 18 ##STR00216##
##STR00217## 0 59 20 ##STR00218## ##STR00219## 1 37
[0302] Inhibition of SCD-1 Activity by
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine Decreases De Novo Fatty Acid Synthesis in CaP Cell
Lines
[0303] The tested compound is the
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine (BZ36).
[0304] It has also been checked that [U--.sup.14C]-palmitate
incorporation into sterol esters, triglycerides and phospholipids,
which is a measure of de novo lipid synthesis was respectively
inhibited by 39%, 27% and 58% in LNCaP (FIG. 4A), and by 33%, 24%
and 41%, respectively in C4-2 cells (FIG. 4B) treated with the
tested compound. Similarly, [U--.sup.14C]-stearate incorporation
into sterol esters, triglycerides and phospholipids was also
inhibited respectively by 77%, 78%, and 81% in LNCaP (FIG. 4D), and
by 70%, 19% and 49%, in C4-2 cells (FIG. 4E), treated with the
tested compound. Similar results were observed in PNT2 cells (FIG.
4C,F).
[0305] SCD1 Inhibition Interferes with Major Signaling Pathways in
Prostate Cancer Cells
[0306] The tested compound is the
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine (BZ36).
[0307] Lipids are important signaling molecules that actively
participate in triggering specific phosphorylation pathways. Since
inhibition of SCD-1 activity was associated with a significant
reduction in de novo lipids synthesis in prostate cancer cells
(FIG. 4), we expected also a decrease in these pathways. The
relative phosphorylation status of several kinases in both LNCaP
and C4-2 cells in response to treatment with the tested compound
(BZ36) showed important differences (FIG. 5A-B). Relevant for our
study was the decrease in Akt phosphorylation (5473/T308) in LNCaP
(FIG. 5A) and C4-2 (FIG. 5B) cells treated with the tested compound
(BZ36), compared to non-treated cells. Significant decreases were
also observed in the phosphorylation of ERK1/2 (T202/Y204,
T185/Y187) and MEK1/2 (S218/5222, S222/S226) in BZ36-treated C4-2
cells as compared to control cells. Interestingly, phosphorylation
of AMPKcc was increased following treatment with the tested
compound (BZ36) in both LNCaP and C4-2 cancer cells (FIG. 5A-B).
Western blot analysis further proved phosphorylation changes in
these proteins (FIG. 5C-D). Inhibition of Akt phosphorylation was
not the result of decreased expression of Akt because total Aid
protein levels were similar in treated and not treated cells (FIG.
5C). In contrast to AKT, treatment with the tested compound (BZ36)
induced a significant increase in phosphorylated AMPKcc in both
LNCaP and C4-2 cells (FIG. 5C). Interestingly, the tested compound
(BZ36) activated AMPK more efficiently than the classical AMPK
activator AICAR (FIG. 5C). We next investigated the active
phosphorylation status of downstream signaling proteins, such as
ERK1/2. Immublot analysis revealed decreased phosphorylation of
ERK1/2 in both LNCaP and C4-2 cells following exposure to the
tested compound (BZ36), whereas total ERK expression was not
changed (FIG. 5D). Moreover, we found that phosphorylation of
GSK3a/13, which is inhibited by phosphorylation by AKT, was also
abrogated by BZ36 treatment in LNCaP and C4-2 cells (FIG. 5D).
These results were consistent with the abrogation of, at least AKT
signaling in cells treated with the tested compound (BZ36).
[0308] AKT is activated by PIP3, which is the result of PIP2
phosphorylation by PI3K. Since SCD-1 inhibition induced a dramatic
decrease in de novo synthesis of phospholipids, which are
PI(3,4,5)P.sub.3 precursors, we anticipated that treatment with the
tested compound (BZ36) would have an impact in PIP3 concentration
in these prostate cancer cells. ELISA test demonstrated that
PI(3,4,5)P.sub.3 concentration was strongly decreased by 84% and
92% respectively in LNCaP and C4-2 BZ36-treated, compared to
non-treated cells. These results suggested that inhibition of AKT
activity in these cells was mediated, at least partially by
decreased synthesis of PI(3,4,5)P.sub.3 precursors after treatment
with the tested compound (BZ36).
[0309] It was also particularly interesting the effects on
GSK3.alpha./.beta., which is further downstream AKT pathway.
Activation of GSK3.alpha./.beta. by SCD-1 inhibitors, resulted in
the disruption of .beta.-catenin signaling, demonstrated by the
decreased activity of a .beta.-cat reporter in response to BZ36 in
both LNCaP, and C4-2 cells (FIG. 5F). This was fully consistent
with changes in the expression of genes in the .beta.-cat pathway,
including, but not limited to decreased expression of several
members of the frizzled family, or decreased cyclin D1, D2, D3,
myc, or c-jun expression, or decreased expression of several Wnt
family members in C4-2 cells treated with the tested compound
(BZ36) (FIG. 5G). Taken together these results proved that SCD-1
inhibition, directly or indirectly results in a strong disruption
of major signaling pathways implicated in cell proliferation,
migration, and survival.
[0310] Inhibition of SCD-1 Activity by
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine Inhibit LNCaP and C4-2 Tumor Growth In Vivo
[0311] The tested compound is the
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
] piperazine (BZ36).
[0312] It has also been evaluated whether reduction of SCD-1
activity could inhibit the growth of CaP cells in mice. LNCaP and
C4-2 cells were injected subcutaneously in male athymic nude mice.
Treatment of each individual mouse started when tumor growth was
measurable. In two independent experiments, it was observed that
treatment of mice with 80 mg/kg of the tested compound
significantly reduced both androgen-dependent LNCaP (FIGS. 6 A-B)
and androgen-insensitive C4-2 (FIGS. 6 D-E) tumor volume and tumor
growth rate, as compared with control mice that received vehicle
only. Moreover, it was observed that these tested compound
treatment at 80 mg/kg induced LNCaP and C4-2 tumor regression in
27% of LNCaP (FIG. 6B) and 19% of C4-2 (FIG. 6E) xenografted mice,
whereas no tumor regression was observed in control mice. On the
opposite, it was observed that LNCaP or C4-2 tumor volume was
increased more than 4-fold in 42% of vehicle-treated mice, whereas
this was the case in only 18% (LNCaP) and 12% (C4-2) of the tested
compound-treated mice (FIGS. 6B, and E). PCNA immunostaining on
tumors from LNCAP (FIG. 6C) and C4-2 (FIG. 6F) xenografts
demonstrated significant decreases of respectively 50% and 41% of
proliferative tumor cells in animals treated with BZ36 at 80 mg/kg
compared to control animals.
[0313] We next examined whether treatment with the tested compound
(BZ36) improved the survival of mice with pre-established
androgen-independent PC tumors derived from C4-2 cells. Nude mice
were treated with vehicle or two doses of BZ36, 80 mg/kg or 160
mg/kg, and followed until death or until tumor volume did reach
2000 mm.sup.3. Among mice bearing subcutaneous C4-2 tumors,
treatment of animals with the tested compound (BZ36) result in
significant and dose-dependant prolongation of animal survival in
comparison to vehicle control (p=0.038, FIG. 7). Indeed, after the
treatment was started, the median survival in the control group was
of 14 days although it was of 21 days in animals treated with 80
mg/kg of the tested compound (BZ36). At 14 days of treatment, 37.5%
of animals did survived in the control group against 75% and 100%
in the groups treated with 80 mg/kg or 160 mg/kg of the tested
compound (BZ36) respectively. At 28 days of treatment, 75% of
animals receiving 160 mg/kg of the tested compound (BZ36) did
survived against only 12.5% in the control group.
[0314] Importantly, no significant weight loss or other toxicity
was observed in nude mice following daily i.p. injection of the
tested compound (BZ36) (FIG. 8A-C). Histological examination
confirmed the absence of toxicity in liver and skin, and no
difference of tissue integrity between animals treated with vehicle
or the tested compound (BZ36) (FIG. 8D).
[0315] Inhibition of SCD-Like Activities in Transformed MEFs
Decreases Proliferation and Tumorigenesis in Mice
[0316] The tested compound is the
1N-[(2-bromo-5-methoxybenzoyl)]-4N-[(phenylpropan)pyridine-3-carboxamide)-
]piperazine (BZ36).
[0317] To further document the strong antiproliferative effects of
SCD inhibitors on Ras-transformed cells we next tested their impact
on Mouse embryonic fibroblasts (MEFs) transformed by genetically
defined elements or genetic background that cooperate with Ras in
transformation. Proliferation index of MEFs fully transformed by
activated Ras (HaRasVl2) and SV40 Large T was strongly decreased by
treatment with the tested compound (BZ36) whereas that of the
slowly growing normal MEFs (WT) was only moderately reduced (FIG.
9A). Similar to prostate cancer cells, inhibition of SCDs activity
in these transformed MEFs resulted in decreased lipogenesis as
measured by the decrease of [.sup.14C]-palmitoleic to palmitic
methyl ester acids ratio (FIG. 9B).
[0318] Importantly, the potent antiproliferative effect of BZ36 on
cells containing Large T confirmed that SCD inhibition has impact
on cells with altered pRB- and p53-pathways, a hallmark of most
cancer cells. Consistently, the tested compound (BZ36) also
efficiently blocked the proliferation of p53-/- MEFs fully
transformed by HaRasVl2 (FIG. 9C). Interestingly, these
antiproliferative effects of the tested compound (BZ36) were
associated with a strong induction of cell death (annexin
V-positive cells) in all transformed cells, including p53-/- MEFS
transformed by activated Ras, suggesting that the end result of SCD
inhibition in cancer cells is a p53-independent cell death (FIG.
9D).
[0319] Investigations have been also conducted to appreciate
whether reduction of SCD-1 activity by the tested compound molecule
would inhibit the development and the growth of HaRasV12-SV40 Large
T-transformed Mefs in vivo.
[0320] When compared with vehicle injection, the tested compound
inhibitor significantly prevented ras SV40 MEFs tumors growth in
mice as early as day 2 after treatment. Moreover, inhibition of
tumor growth by the tested compound was maintained all along the 12
days of this experiment (FIG. 9E) This experiment proved that this
treatment was efficient in the presence of activated Ras, which is
a common situation found in human cancers. PCNA immunostaining on
tumors from ras SV40 MEFs xenografts demonstrated a significant 61%
decrease of proliferative tumor cells in mice treated with the
tested compound, compared to vehicle-treated mice (FIG. 9F).
[0321] Furthermore, it has also been shown that pre-treatment of
mice with the tested compound one week prior tumor generation by
ras SV40 MEFs, reduced tumor formation. Five days after cells were
grafted, almost all of vehicle treated mice developed tumors,
whereas tumors grown only in 50% of the tested compound treated
mice (FIG. 9G). This suggested that Ras-mediated transformation
requires active de novo lipid synthesis in order to generate
cancers. Collectively, these data confirmed our observation with
LNCaP and C4-2 human cancer cell lines, and demonstrate that
inhibition of SCD activity has a potent inhibitory effect on the
cell viability and tumorigenicity of p53-deficient and
Ras-transformed cells.
[0322] Accordingly, the compounds according to the instant
invention and more particularly compound of formula (I) are
particularly useful for the treatment of prostate cancer.
[0323] According to the present invention, such compound of formula
I could be particularly useful in combination with standard
therapy, in any clinical situation in which a strong response is
expected from standard therapy, and in which, nevertheless,
relapses are frequent.
[0324] By "another anti-cancer treatment" is meant any other
suitable treatment approved for cancer treatment. In particular,
said other anti-cancer treatment may be selected from the group
consisting of chemotherapy, surgery, radiotherapy, hormonotherapy,
and/or immunotherapy.
[0325] In the case of chemotherapy, at least one secondary
chemotherapeutic agent may be used. Such an agent may be selected
from the group consisting of growth inhibitory agents (defined as
compounds or compositions which inhibit growth of a cell, either in
vitro or in vivo) and cytotoxic agents (defined as compounds or
compositions which inhibits or prevents the function of cells
and/or causes destruction of cells).
[0326] In a particular embodiment of the invention, a secondary
chemotherapeutic agent may be selected from the group consisting of
taxanes, topoisomerase II inhibitors, DNA alkylating agents,
anti-metabolite, anti-tubuline, vinca alkaloids, intercalating
agents and platinium salts.
[0327] In a more particular embodiment of the invention, a
secondary chemotherapeutic agent may be selected from the group
consisting of: paclitaxel, docetaxel, doxorubicin, epirubicin,
daunorubicin, etoposide, bleomycin, tamoxifen, prednisone,
dacarbazine, mechlorethamine, methotrexate, 5-fluorouracil,
anthracyclines, adriamicin, vinblastine, vincristine, vinorelbine,
topotecan, carboplatin, cisplatin, permetrexed, irinotecan,
gemcitabine, gefinitib, erlotinib, fludarabin, ifosfamide,
procarbazine, mitoxanthrone, melphalan, mitomycin C, chlorambucil,
cyclophosphamide and platinium salts.
[0328] Of course, any suitable combination of chemotherapeutic
drugs may be used, depending on the type of cancer.
[0329] Compounds of formule IIa), pharmaceutical compositions and
therapeutic methods based on such compounds are particularly useful
for the treatment and/or prevention of diseases mediated by
stearoyl-CoA desaturase (SCD), especially human SCD (hSCD), by
administering to a patient in need of such treatment an effective
amount of an SCD-inhibiting, agent.
[0330] An SCD-mediated disease or condition also includes metabolic
syndrome (including but not limited to dyslipidemia, obesity and
insulin resistance, hypertension, microalbuminemia, hyperuricaemia,
and hypercoagulability), Syndrome X, diabetes, insulin resistance,
decreased glucose tolerance, non-insulin-dependent diabetes
mellitus, Type II diabetes, Type I diabetes, diabetic
complications, body weight disorders, weight loss, body mass index
and leptin related diseases.
[0331] As used herein, the term "metabolic syndrome" is a
recognized clinical term used to describe a condition comprising
combinations of Type II diabetes, impaired glucose tolerance,
insulin resistance, hypertension, obesity, increased abdominal
girth, hypertriglyceridemia, low HDL, hyperuricaemia,
hypercoagulability and/or microalbuminemia.
[0332] An SCD-mediated disease or condition also includes fatty
liver, hepatic steatosis, hepatitis, non-alcoholic hepatitis,
non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute
fatty liver, fatty liver of pregnancy, drug-induced hepatitis,
erythrohepatic protoporphyria, iron overload disorders, hereditary
hemochromatosis, hepatic fibrosis, hepatic cirrhosis, hepatoma and
conditions related thereto.
[0333] An SCD-mediated disease or condition also includes but is
not limited to a disease or condition which is, or is related to
primary hypertriglyceridemia, or hypertriglyceridemia secondary to
another disorder or disease, such as hyperlipoproteinemias,
familial histiocytic reticulosis, lipoprotein lipase deficiency,
apolipoprotein deficiency (such as ApoCII deficiency or ApoE
deficiency), and the like, or hypertriglyceridemia of unknown or
unspecified ethio logy.
[0334] An SCD-mediated disease or condition also includes a
disorder of polyunsaturated fatty acid (PUFA) disorder, or a skin
disorder, including but not limited to eczema, acne, psoriasis,
keloid scar formation or prevention, diseases related to production
or secretions from mucous membranes, such as monounsaturated fatty
acids, wax esters, and the like. An SCD-mediated disease or
condition also includes inflammation, sinusitis, asthma,
pancreatitis, osteoarthritis, rheumatoid arthritis, cystic
fibrosis, and pre-menstrual syndrome.
[0335] An SCD-mediated disease or condition also includes but is
not limited to a disease or condition which is, or is related to
cancer, more particularly lung cancer and prostate cancer, breast
cancer, hepatomas and the like, neoplasia, malignancy, metastases,
tumours (benign or malignant), carcinogenesis.
[0336] An SCD-mediated disease or condition also includes a
condition where increasing lean body mass or lean muscle mass is
desired, such as is desirable in enhancing performance through
muscle building. Myopathies and lipid myopathies such as carnitine
palmitoyltransferase deficiency (CPT I or CPT II) are also included
herein. Such treatments are useful in humans and in animal
husbandry, including for administration to bovine, porcine or avian
domestic animals or any other animal to reduce triglyceride
production and/or provide leaner meat products and/or healthier
animals.
[0337] An SCD-mediated disease or condition also includes a disease
or condition which is, or is related to, neurological diseases,
psychiatric disorders, multiple sclerosis, eye diseases, and immune
disorders.
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